Bispecific antibody fusion molecules and methods of use thereof

Abstract

Described herein are compositions and methods for efficient production of heteromultimeric antibodies, such as bispecific antibodies. The compositions and methods improve the assembly of heteromultimeric antibodies with higher yield and efficiency than conventional methods. The antibodies are optionally fused to a cytokine or costimulatory peptide or portion thereof. Also described herein are monoclonal antibodies and antigen-binding fragments thereof, variants, multimeric forms, or bispecific antibodies that specifically bind to CD3. Described herein are methods for making and using anti-CD3 antibodies and antigen-binding fragments thereof in various therapeutic, diagnostic, and prophylactic applications.

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 368,852, filed July 19, 2022, U.S. Provisional Application No. 63 / 330,250, filed April 12, 2022, and U.S. Provisional Application No. 63 / 321,563, filed March 18, 2022, the contents of each of which are incorporated by reference in their entirety into this specification.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING The contents of the electronic sequence listing entitled "EVIM_001_001WO_SeqList_ST26.xml", created on March 20, 2023 and having a size of 883,743 bytes, are incorporated herein by reference in their entirety.

[0003] Described herein are compositions and methods for the efficient production of antibodies or antigen-binding fragments thereof. Antibodies may be capable of specifically binding to multiple target molecules, or different epitopes on a single target molecule. The present compositions and methods improve the assembly and production of antibodies, resulting in high efficiency and yields, as compared to conventional methods. [Background technology]

[0004] Monoclonal antibodies of the IgG type contain two identical antigen-binding arms and a constant domain (Fc). Antibodies with different specificities of the binding arms usually do not occur in nature and must be produced using chemical engineering (e.g., chemical cross-linking), recombinant DNA and / or cell fusion techniques.

[0005] Bispecific antibodies are able to bind two different antigens simultaneously. This property allows the development of therapeutic strategies not possible with conventional monoclonal antibodies. Another class of multispecific molecules is recombinant fusion proteins. Recombinant fusion proteins consisting of the extracellular domain of an immunomodulatory protein and the constant (Fc) domain of an immunoglobulin (Ig) represent an evolving class of human therapeutics.

[0006] The production of clinical grade material continues to be challenging for antibodies in general, and for multispecific antibodies in particular. Many routes exist for the production of molecules with mixed binding arms, i.e., binding arms that are not identical to each other. However, each of these methods has significant drawbacks.

[0007] Chemical cross-linking is labor intensive because it may require additional steps to separate undesired products from desired products, requiring further purification of related species from homodimers and other undesired by-products. Furthermore, chemical modification steps may alter protein integrity, reducing stability. Therefore, this method is often inefficient and may lead to loss of antibody activity.

[0008] Cell fusion techniques (e.g., hybrid hybridomas) express two heavy chains and two light chains that randomly assemble, and when two cells, each expressing a separate antibody, are fused, 10 different antibody combinations are generated. The desired heteromultimeric antibody is only a small fraction of the antibodies thus produced. Purification of the desired heteromultimeric protein dramatically reduces production yields and increases manufacturing costs.

[0009] Using recombinant DNA techniques, various heteromultimeric formats have been generated, such as single chain Fvs, diabodies, etc. that do not contain an Fc domain. The major drawback of this type of antibody molecule is the lack of an Fc domain, which eliminates the antibody's ability to elicit effector functions and extend serum half-life (e.g., complement activation, Fc receptor binding, etc.). Therefore, bispecific antibodies that contain a functional Fc domain are desirable.

[0010] Recombinant DNA techniques have also been used to generate "knobs-into-holes" bispecific antibodies. One limitation of this strategy is that the light chains of the two parent antibodies must be identical to prevent mispairing and the formation of unwanted and / or inactive molecules when expressed in the same cell.

[0011] Furthermore, one of the limiting events during annealing and purification is the redox efficiency. Following reduction, the oxidized heterodimer typically constitutes only 70-80% of the protein after this step, as shown by BioAnalyzer and MS-TOF analysis of the intact mass species after oxidation. The remaining 20-30% of the antibody is dimeric and lacks covalent bonds (SEC-MALS). This can be removed, but will greatly affect the overall yield.

[0012] Thus, there remains a need to improve the overall yield in antibody production, particularly heterodimeric antibodies such as bispecific antibodies. Described herein are methods that may improve the overall yield of bispecific antibodies, heterodimers, etc. These and other aspects and advantages of the invention will become apparent from the description of the invention provided herein. Summary of the Invention

[0013] The present disclosure provides an antibody having the following structure: a. a variable region (VH1), and a constant region 1 domain (CH1 H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domains (CH1 H3a. a first heavy chain polypeptide (H1) comprising a constant region (CH1) having a variable region (VL1) and a constant region (CL1); b. a first light chain polypeptide (L1) comprising a variable region (VH2), and a constant region 1 domain (CH2 H1 ), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3 and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2), wherein i. the amino acid at position 39 (Kabat numbering) of VH1 and VH2 is a charged or polar amino acid residue and the amino acid at position 38 (Kabat numbering) of VL1 and VL2 is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 39 of VH1 and VH2, or the amino acid at position 100 (Kabat numbering) of VH1 and VH2 is a charged or polar amino acid residue and the amino acid at position 44 (Kabat numbering) of VL1 and VL2 is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 100 (Kabat numbering) of VH1 and VH2, and ii. H1 and CH1 H2 The amino acid at position 147 of CL1 or CL2 (EU numbering) is a charged or polar amino acid residue, and one of the amino acids at positions 131, 179 or 180 of CL1 or CL2 (EU numbering) is a CH1 H1 and CH1 H2 an amino acid residue having an opposite charge or polarity compared to the amino acid at position 147 (EU numbering) of H1 and CH1 H2 The amino acid at position 185 (EU numbering) of CL1 is a charged or polar amino acid residue, and the amino acid at position 137 (EU numbering) of CL1 and CL2 is a CH1 H1 and CH1 H2 or an amino acid residue having an opposite charge or polarity compared to the amino acid at position 185 (EU numbering) of CH1 H1 and CH1 H2The amino acid at position 187 (EU numbering) of CL1 is a charged or polar amino acid residue, and one of the amino acids at positions 137 or 138 (EU numbering) of CL1 and CL2 is a CH1 H1 and CH1 H2 an amino acid residue having an opposite charge or polarity compared to the amino acid at position 187 (EU numbering) of H1 and CH1 H2 The amino acid at position 145 (EU numbering) of CL1 is a charged or polar amino acid residue, and the amino acid at position 131 (EU numbering) of CL1 and CL2 is a CH1 H1 and CH1 H2 The antibody provides an antibody in which the amino acid residue at position 145 (EU numbering) of is an amino acid residue having an opposite charge or polarity compared to the amino acid at position 145 (EU numbering).

[0014] In some embodiments, the charged amino acid residue is a naturally occurring or non-naturally occurring amino acid, hi some embodiments, the naturally occurring charged amino acid residue is arginine, lysine, histidine, glutamic acid, or aspartic acid.

[0015] In some embodiments, the H1 amino acids at positions 39, 100, 147, 185, 187, or 145 are positively charged, the L1 amino acids at positions 38, 44, 131, 179, 180, 137, or 138 are negatively charged, the H2 amino acids at positions 39, 100, 147, 185, 187, or 145 are negatively charged, and the L2 amino acids at positions 38, 44, 131, 179, 180, 137, or 138 are positively charged. In some embodiments, the H1 amino acids at positions 39, 100, 147, 185, 187, and 145 are negatively charged, the L1 amino acids at positions 38, 44, 131, 179, 180, 137, or 138 are positively charged, the H2 amino acids at positions 39, 100, 147, 185, 187, or 145 are positively charged, and the L2 amino acids at positions 38, 44, 131, 179, 180, 137, or 138 are negatively charged.

[0016] In some embodiments, L1 and L2 are lambda or kappa light chains. In some embodiments, L1 is a lambda light chain and L2 is a kappa light chain. In some embodiments, L1 is a kappa light chain and L2 is a lambda light chain.

[0017] In some embodiments, the antibody is an IgG 1 , IgG 2 , or IgG 4 In some embodiments, the antibody is a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, the antibody is a bispecific antibody.

[0018] In some embodiments, the bispecific antibody comprises i) a first antigen-binding domain that binds to a cell surface antigen, where the cell surface antigen is expressed on a T cell, a NK cell, a neutrophil, a B cell, or a dendritic cell engager, and ii) a second antigen-binding domain that binds to a disease-associated antigen (DAA). In some embodiments, the cell surface antigen is expressed on a T cell.

[0019] In some embodiments, the cell surface antigen expressed on T cells is CD3. In some embodiments, the cell surface antigen expressed on T cells is CD3ε.

[0020] In some embodiments, the DAA is UL16 binding protein 2 (ULBP2). In some embodiments, the DAA is UL16 binding protein 5 (ULBP5). In some embodiments, the DAA is UL16 binding protein 6 (ULBP6).

[0021] In some embodiments, the polypeptide is fused to the N-terminus or C-terminus of H1 and / or H2. In some embodiments, the polypeptide is fused to the N-terminus of H1. In some embodiments, the polypeptide is fused to the N-terminus of H2. In some embodiments, the polypeptide is fused to the C-terminus of H1. In some embodiments, the polypeptide is fused to the C-terminus of H2. In some embodiments, the polypeptide is CD58, IL-7, or a fragment thereof. In some embodiments, the polypeptide is the CD58 extracellular domain (CD58-ECD). In some embodiments, the polypeptide is the CD58 variable domain (CD58v * ).

[0022] In some embodiments, the polypeptides are fused via a linker peptide. In some embodiments, the linker peptide is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acid residues in length. In some embodiments, the linker peptide comprises the amino acid sequence of SEQ ID NO: 52-54.

[0023] In some embodiments, the antibodies include i) those in which the amino acid at position 87 (Kabat numbering) of VH1 and / or VH2 is G, and ii) those in which the amino acid at position 45 (Kabat numbering) of VL1 and / or VL2 is W.

[0024] In some embodiments, the antibody comprises i) CH1 H3 has C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering), CH2 H3 has C at position 354 and W at position 366 (EU numbering), ii) CH2 H3 has C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering), CH1 H3has C at position 354 and W at position 366 (EU numbering), iii) CH1 H3 has C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering), CH2 H3 has a C at position 349 and a W at position 366 (EU numbering), or iv) CH2 H3 has C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering), CH1 H3 However, this includes those with a C at position 349 and a W at position 366 (EU numbering).

[0025] In some embodiments, CH1 H3 and / or CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0026] In some embodiments, i) H1H and / or H2H have A at positions 234 and 235 (EU numbering); ii) H1H and / or H2H have A at positions 234, 235 and 237 (EU numbering); or iii) H1H and / or H2H have A at positions 234 and 235 and G at position 329 (EU numbering).

[0027] In some embodiments, the antibody comprises i) CH1 H3 and / or CH2 H3 has A at position 297 (EU numbering), ii) CH1 H3 and / or CH2 H3 or iii) CH1 H3 and / or CH2 H3 but with S at position 297 (EU numbering). H3 and / or CH2 H3 has S at position 331 (EU numbering).

[0028] In some embodiments, the antibody comprises i) CH1 H2 and / or CH2 H2 but has C at position 370 (Kabat numbering), and ii) CH1 H2and / or CH2 H2 However, some have a C at position 375 (Kabat numbering).

[0029] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. H1 and the amino acid at position 137 (EU numbering) of CL1 is D; H1 in which the amino acid at position 128 (EU numbering) in VH2 is C and the amino acid at position 118 (EU numbering) in CL1 is C; and iv. in which the amino acid at position 220 (EU numbering) in H1H is S and the amino acid at position 214 (EU numbering) in CL1 is S; b) H2 and L2 include: i. in VH2 the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; and ii. in CH2 H1 and the amino acid at position 180 (EU numbering) of CL2 is R.

[0030] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii. H1 in which the amino acid at position 185 (EU numbering) is K and in CL1 the amino acid at position 137 (EU numbering) is D; b) H2 and L2 are as follows: i. VH2 in which the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; ii. CH2 H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1and iv.H2H has an amino acid at position 134 (EU numbering) that is C and CL2 has an amino acid at position 116 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0031] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii. H1 in which the amino acid at position 185 (EU numbering) is K and in CL1 the amino acid at position 137 (EU numbering) is D; b) H2 and L2 are as follows: i. VH2 in which the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; ii. CH2 H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1 and iv.H2H has an amino acid at position 136 (EU numbering) that is C and CL2 has an amino acid at position 114 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0032] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. H1 and CL1, wherein the amino acid at position 147 (EU numbering) is K and the amino acid at position 131 (EU numbering) of CH1 is D; H1in which the amino acid at position 173 (EU numbering) in VH2 is C and the amino acid at position 162 (EU numbering) in CL1 is C; iv. in which the amino acid at position 220 (EU numbering) in H1H is S and the amino acid at position 214 (EU numbering) in CL1 is S; b) H2 and L2 include: i. in VH2 the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; and ii. H1 and the amino acid at position 180 (EU numbering) of CL2 is R.

[0033] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. H1 and the amino acid at position 137 (EU numbering) of CL1 is D; H1 in which the amino acid at position 173 (EU numbering) in VH2 is C and the amino acid at position 162 (EU numbering) in CL1 is C; and iv. in which the amino acid at position 220 (EU numbering) in H1H is S and the amino acid at position 214 (EU numbering) in CL1 is S; b) H2 and L2 include: i. in VH2 the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; and ii. in CH2 H1 and the amino acid at position 180 (EU numbering) of CL2 is R.

[0034] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii. H1in which the amino acid at position 185 (EU numbering) is K and in CL1 at position 137 (EU numbering) is D; b) H2 and L2 are as follows: i. in VH2 the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; ii. H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1 and iv.H2H has an amino acid at position 131 (EU numbering) that is C and CL2 has an amino acid at position 114 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0035] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii. H1 in which the amino acid at position 185 (EU numbering) is K and in CL1 the amino acid at position 137 (EU numbering) is D; b) H2 and L2 are as follows: i. VH2 in which the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; ii. CH2 H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv.H2H has an amino acid at position 170 (EU numbering) that is C and CL2 has an amino acid at position 162 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0036] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. H1i. the amino acid at position 185 (EU numbering) of VH2 is E and the amino acid at position 137 (EU numbering) of CL1 is K, and iii. the amino acid at position 179 (EU numbering) of CL1 is E; b) H2 and L2 include: i. the amino acid at position 39 (Kabat numbering) of VH2 is D and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv.H2H has an amino acid at position 171 (EU numbering) that is C and CL2 has an amino acid at position 162 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0037] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii. H1 in which the amino acid at position 185 (EU numbering) is K and in CL1 the amino acid at position 137 (EU numbering) is D; b) H2 and L2 are as follows: i. VH2 in which the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; ii. CH2 H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv.H2H has an amino acid at position 171 (EU numbering) that is C and CL2 has an amino acid at position 162 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0038] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. H1 i. the amino acid at position 185 (EU numbering) of VH2 is E and the amino acid at position 137 (EU numbering) of CL1 is K, and iii. the amino acid at position 179 (EU numbering) of CL1 is E; b) H2 and L2 include: i. the amino acid at position 39 (Kabat numbering) of VH2 is D and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1 and iv.H2H has an amino acid at position 171 (EU numbering) that is C and CL2 has an amino acid at position 162 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0039] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii. H1 in which the amino acid at position 185 (EU numbering) is K and in CL1 the amino acid at position 137 (EU numbering) is D; b) H2 and L2 are as follows: i. VH2 in which the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; ii. CH2 H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1 and iv.H2H has an amino acid at position 171 (EU numbering) that is C and CL2 has an amino acid at position 162 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0040] In some embodiments, a) H1 and L1 are as follows: VH1, the amino acid at position 39 (Kabat numbering) is K, and VL1, the amino acid at position 38 (Kabat numbering) is D; CH1 H1 and CL1, in which the amino acid at position 147 (EU numbering) is K, and CL1, in which the amino acid at position 131 (EU numbering) is D; H1 and in which the amino acid at position 185 (EU numbering) of CL1 is K and the amino acid at position 137 (EU numbering) of CL1 is D; b) H2 and L2 include the following: VH2 has an amino acid at position 39 (Kabat numbering) that is D and VL2 has an amino acid at position 38 (Kabat numbering) that is K; CH2 H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2, H1 and the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0041] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is D and the amino acid at position 38 (Kabat numbering) of VL1 is K; and ii. H1 in which the amino acid at position 185 (EU numbering) of VH2 is E and in which the amino acid at position 137 (EU numbering) of CL1 is K; b) H2 and L2 are as follows: i. in VH2 the amino acid at position 39 (Kabat numbering) is K and in VL2 the amino acid at position 38 (Kabat numbering) is D; ii. H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1and iv.H2H has an amino acid at position 136 (EU numbering) that is C and CL2 has an amino acid at position 114 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0042] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is D and the amino acid at position 38 (Kabat numbering) of VL1 is K; and ii. H1 in which the amino acid at position 185 (EU numbering) of VH2 is E and in which the amino acid at position 137 (EU numbering) of CL1 is K; b) H2 and L2 are as follows: i. in VH2 the amino acid at position 39 (Kabat numbering) is K and in VL2 the amino acid at position 38 (Kabat numbering) is D; ii. H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv.H2H has an amino acid at position 171 (EU numbering) that is C and CL2 has an amino acid at position 162 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0043] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is D and the amino acid at position 38 (Kabat numbering) of VL1 is K; and ii. H1 in which the amino acid at position 185 (EU numbering) is K and in CL1 the amino acid at position 137 (EU numbering) is D; b) H2 and L2 are as follows: i. VH2 in which the amino acid at position 39 (Kabat numbering) is K and in VL2 the amino acid at position 38 (Kabat numbering) is D; ii. CH2 H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1and iv.H2H has an amino acid at position 171 (EU numbering) that is C and CL2 has an amino acid at position 162 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0044] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is D and the amino acid at position 38 (Kabat numbering) of VL1 is K; ii. CH1 H1 and CL1, wherein the amino acid at position 147 (EU numbering) is K and the amino acid at position 131 (EU numbering) of CH1 is D; H1 and iv. CH1 H1 in which the amino acid at position 145 (EU numbering) of VH2 is S and in which the amino acid at position 180 (EU numbering) of CL1 is E; b) H2 and L2 are as follows: i. in VH2 the amino acid at position 39 (Kabat numbering) is K and in VL2 the amino acid at position 38 (Kabat numbering) is D; ii. H1 in which the amino acid at position 187 (EU numbering) in VH2 is D and the amino acid at position 138 (EU numbering) in CL2 is K; iii. in which the amino acid at position 170 (EU numbering) in VH2 is C and the amino acid at position 162 (EU numbering) in VL2 is C; and iv. in which the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0045] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. H1 and iii. CH1 H1in which the amino acid at position 185 (EU numbering) of VH2 is E and in which the amino acid at position 137 (EU numbering) of CL1 is D; b) H2 and L2 are as follows: i. in VH2 the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; ii. H1 and the amino acid at position 137 (EU numbering) of CL2 is K; iii. the amino acid at position 138 (EU numbering) of CL2 is R; iv. H1 and v.H2H where the amino acid at position 170 (EU numbering) is C and v.H2H where the amino acid at position 162 (EU numbering) is C; and v.H2H where the amino acid at position 220 (EU numbering) is S and v.H2H where the amino acid at position 214 (EU numbering) is S.

[0046] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. H1 and iii. CH1 H1 in which the amino acid at position 145 (EU numbering) is S; and b) H2 and L2 are as follows: i. in VH2 the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; ii. H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv.H2H has an amino acid at position 170 (EU numbering) that is C and CL2 has an amino acid at position 162 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0047] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is D and the amino acid at position 38 (Kabat numbering) of VL1 is K; ii. CH1 H1 and iii. CH1 H1 in which the amino acid at position 185 (EU numbering) of VH2 is E and in which the amino acid at position 137 (EU numbering) of CL1 is K; b) H2 and L2 are as follows: i. in VH2 the amino acid at position 39 (Kabat numbering) is K and in VL2 the amino acid at position 38 (Kabat numbering) is D; ii. H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1 and iv.H2H has an amino acid at position 136 (EU numbering) that is C and CL2 has an amino acid at position 114 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0048] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is E; ii. CH1 H1 and iii. CH1 H1 in which the amino acid at position 185 (EU numbering) of VH2 is E and in which the amino acid at position 137 (EU numbering) of CL1 is K; b) H2 and L2 are as follows: i. in VH2 the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; ii. H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1and iv.H2H has an amino acid at position 136 (EU numbering) that is C and CL2 has an amino acid at position 114 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0049] In some embodiments, a) H1 and L1 are as follows: i. the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. H1 and iii. CH1 H1 in which the amino acid at position 185 (EU numbering) is D and in CL1 the amino acid at position 137 (EU numbering) is K; b) H2 and L2 are as follows: i. VH2 in which the amino acid at position 39 (Kabat numbering) is D and in VL2 the amino acid at position 38 (Kabat numbering) is K; ii. CH2 H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv.H2H has an amino acid at position 171 (EU numbering) that is C and CL2 has an amino acid at position 162 (EU numbering) that is C; and iv.H2H has an amino acid at position 220 (EU numbering) that is S and CL2 has an amino acid at position 214 (EU numbering) that is S.

[0050] In some embodiments, i) the amino acid at position 87 (Kabat numbering) of VH1 and / or VH2 is G and ii) the amino acid at position 45 (Kabat numbering) of VL1 and / or VL2 is W.

[0051] In some embodiments, i) CH1 H3 has C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering), and CH2 H3 has C at position 354 and W at position 366 (EU numbering), ii) CH2H3 has C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering), and CH1 H3 has C at position 354 and W at position 366 (EU numbering), iii) CH1 H3 has C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering), and CH2 H3 has a C at position 349 and a W at position 366 (EU numbering), or iv) CH2 H3 has C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering), and CH1 H3 has a C at position 349 and a W at position 366 (EU numbering). H3 and / or CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0052] In some embodiments, i) H1H and / or H2H have A at positions 234 and 235 (EU numbering); ii) H1H and / or H2H have A at positions 234, 235 and 237 (EU numbering); or iii) H1H and / or H2H have A at positions 234 and 235 and G at position 329 (EU numbering).

[0053] In some embodiments, i) CH1 H3 and / or CH2 H3 has A at position 297 (EU numbering), ii) CH1 H3 and / or CH2 H3 has G at position 297 (EU numbering), or iii) CH1 H3 and / or CH2 H3 has an S at position 297 (EU numbering). H3 and / or CH2 H3 has S at number 331 (EU numbering).

[0054] In some embodiments, the antibody comprises i) CH1 H2 and / or CH2 H2but has C at position 370 (Kabat numbering), and ii) CH1 H2 and / or CH2 H2 However, some have a C at position 375 (Kabat numbering).

[0055] In some embodiments, a) VH1 comprises the amino acid sequence of SEQ ID NO:13, and b) VL1 comprises the amino acid sequence of SEQ ID NO:22.

[0056] In some embodiments, a) VH1 is [ka] wherein X1 of SEQ ID NO:440 is D or T, X2 of SEQ ID NO:440 is D or N, X3 of SEQ ID NO:440 is D or K, X4 of SEQ ID NO:440 is K or R, X5 of SEQ ID NO:440 is N or T, X6 of SEQ ID NO:440 is D or N, and X7 of SEQ ID NO:440 is D or S; and b) VL1 is [ka] wherein X1 of SEQ ID NO:441 is E or D, X2 of SEQ ID NO:441 is D or G, X3 of SEQ ID NO:441 is D or Y, and X4 of SEQ ID NO:441 is D, N, or Q.

[0057] In some embodiments, a) VH2 comprises a complementarity determining region 1 (VH2) comprising the amino acid sequence of SEQ ID NO: 428. CDR1 ), a complementarity determining region 2 (VH2) comprising the amino acid sequence of SEQ ID NO: 430 CDR2 ), and a complementarity determining region 3 (VH2) comprising the amino acid sequence of SEQ ID NO: 432 CDR3 ), and b) VL2 comprises a complementarity determining region 1 (VL2) comprising the amino acid sequence of SEQ ID NO: 433. CDR1 ), complementarity determining region 2 (VL2) comprising the amino acid sequence of SEQ ID NO:434 CDR2 ), and a complementarity determining region 3 (VL2CDR3 ).

[0058] In some embodiments, a) VH2 comprises a complementarity determining region 1 (VH2) comprising the amino acid sequence of SEQ ID NO:5. CDR1 ), a complementarity determining region 2 (VH2) comprising the amino acid sequence of SEQ ID NO:7 CDR2 ), and a complementarity determining region 3 (VH2) comprising the amino acid sequence of SEQ ID NO:9 CDR3 ) and b) VL2 comprises a complementarity determining region 1 (VL2) comprising the amino acid sequence of SEQ ID NO: 10. CDR1 ), complementarity determining region 2 (VL2) comprising the amino acid sequence of SEQ ID NO:11 CDR2 ), and a complementarity determining region 3 (VL2 CDR3 ).

[0059] In some embodiments, CD58 comprises the amino acid sequence of SEQ ID NO: 49 to 50. In some embodiments, IL-7 comprises the amino acid sequence of SEQ ID NO: 51.

[0060] The present disclosure provides a polynucleotide comprising a nucleic acid sequence encoding any one of the antibodies disclosed herein. The present disclosure also provides a vector comprising the polynucleotide of the present disclosure.

[0061] The present disclosure also provides a pharmaceutical composition comprising any one of the antibodies, polynucleotides, or vectors of the present disclosure and a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical composition is packaged in a liposome or lipid nanoparticle.

[0062] The present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering any one of the pharmaceutical compositions of the present disclosure in a therapeutically effective amount.The present disclosure also provides a method for retargeting T cells in a subject in need thereof, comprising administering any one of the pharmaceutical compositions of the present disclosure in a therapeutically effective amount.The present disclosure also provides a method for activating T cells in a subject in need thereof, comprising administering any one of the pharmaceutical compositions of the present disclosure in a therapeutically effective amount.

[0063] In some embodiments, the subject has cancer. In some embodiments, the cancer is a ULBP2 positive cancer. In some embodiments, the cancer is a primary tumor, a metastatic cancer, a multidrug resistant cancer, an advanced tumor, or a recurrent cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a urothelial cancer, a lung cancer, a brain cancer, a head and neck cancer, a breast cancer, a skin cancer, a melanoma, a liver cancer, a pancreatic cancer, a gastric cancer, a colon cancer, a rectal cancer, a uterine cancer, a cervical cancer, an ovarian cancer, a prostate cancer, a testicular cancer, a skin cancer, or an esophageal cancer.

[0064] In some embodiments, the method further comprises administering a therapeutically effective amount of a ligand or cytokine, or an agonist antibody that binds to a ligand and a receptor for a cytokine. In some embodiments, the ligand is CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, ICOSLG, MICA, MICB, ULBP1, ULBP2, ULBP3, RAET1G, and RAET1L. In some embodiments, the cytokine is IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, or IL-21.

[0065] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0066] [Figure 1A]Schematic diagrams of antibodies with charge pair mutations, disulfide bond rearrangements, and knob-into-hole mutations are shown. The domains in grey represent a first heavy chain polypeptide (H1) having a heavy chain variable region (VH1) with a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2), and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1). The domains in white represent a second heavy chain polypeptide (H2) having a variable region (VH2) and a constant region (CH2) with a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2), and a constant region 3 domain (CH2H3); and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2). The + and - symbols between the antigen-binding domains represent charge pair mutations. The lines between the CH1H1 and CL1 domains and between the CH2H1 and CL2 domains represent disulfide bonds, the solid lines represent intrinsic disulfide bonds, and the dashed lines represent rearranged disulfide bonds. The convex and concave portions between the CH1H3 and CH2H3 domains represent knob-into-hole mutations. The charge pair mutations, disulfide bond rearrangements, and knob-into-hole mutations result in increased heterodimerization of the heavy and light chains, which is advantageous for the production and purification of the bispecific antibodies of the present disclosure. [Figure 1B]Schematic diagrams of antibodies with charge pair mutations, disulfide bond rearrangements, and knob-into-hole mutations are shown. The domains in grey represent a first heavy chain polypeptide (H1) having a heavy chain variable region (VH1) with a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2), and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1). The domains in white represent a second heavy chain polypeptide (H2) having a variable region (VH2) and a constant region (CH2) with a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2), and a constant region 3 domain (CH2H3); and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2). The + and - symbols between the antigen-binding domains represent charge pair mutations. The lines between the CH1H1 and CL1 domains and between the CH2H1 and CL2 domains represent disulfide bonds, the solid lines represent intrinsic disulfide bonds, and the dashed lines represent rearranged disulfide bonds. The convex and concave portions between the CH1H3 and CH2H3 domains represent knob-into-hole mutations. The charge pair mutations, disulfide bond rearrangements, and knob-into-hole mutations result in increased heterodimerization of the heavy and light chains, which is advantageous for the production and purification of the bispecific antibodies of the present disclosure. [Figure 1C]Schematic diagrams of antibodies with charge pair mutations, disulfide bond rearrangements, and knob-into-hole mutations are shown. The domains in grey represent a first heavy chain polypeptide (H1) having a heavy chain variable region (VH1) with a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2), and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1). The domains in white represent a second heavy chain polypeptide (H2) having a variable region (VH2) and a constant region (CH2) with a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2), and a constant region 3 domain (CH2H3); and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2). The + and - symbols between the antigen-binding domains represent charge pair mutations. The lines between the CH1H1 and CL1 domains and between the CH2H1 and CL2 domains represent disulfide bonds, the solid lines represent intrinsic disulfide bonds, and the dashed lines represent rearranged disulfide bonds. The convex and concave portions between the CH1H3 and CH2H3 domains represent knob-into-hole mutations. The charge pair mutations, disulfide bond rearrangements, and knob-into-hole mutations result in increased heterodimerization of the heavy and light chains, which is advantageous for the production and purification of the bispecific antibodies of the present disclosure. [Figure 1D]Schematic diagrams of antibodies with charge pair mutations, disulfide bond rearrangements, and knob-into-hole mutations are shown. The domains in grey represent a first heavy chain polypeptide (H1) having a heavy chain variable region (VH1) with a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2), and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1). The domains in white represent a second heavy chain polypeptide (H2) having a variable region (VH2) and a constant region (CH2) with a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2), and a constant region 3 domain (CH2H3); and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2). The + and - symbols between the antigen-binding domains represent charge pair mutations. The lines between the CH1H1 and CL1 domains and between the CH2H1 and CL2 domains represent disulfide bonds, the solid lines represent intrinsic disulfide bonds, and the dashed lines represent rearranged disulfide bonds. The convex and concave portions between the CH1H3 and CH2H3 domains represent knob-into-hole mutations. The charge pair mutations, disulfide bond rearrangements, and knob-into-hole mutations result in increased heterodimerization of the heavy and light chains, which is advantageous for the production and purification of the bispecific antibodies of the present disclosure. [Diagram 2] (A-B) are two graphs showing the biophysical characterization of light chain paired bispecific antibodies EIP0187 (light chain pairing C), EIP0205 (light chain pairing D), EIP0356 (light chain pairing O) and EIP0377 (light chain pairing P) compared to the EIP0112 Crossmab control antibody. (A) is a size exclusion chromatogram obtained from tandem purification of light chain paired bispecific antibodies by Protein A and size exclusion chromatography. (B) shows differential scanning calorimetry analysis of light chain paired bispecific antibodies. [Diagram 3]A-B are NuPAGE gel analyses of representative variants of light chain paired bispecific antibodies shown in Figures 2A-2B. A is a non-reducing NuPAGE analysis. B is a reducing NuPAGE analysis. Together, these show an intact bispecific antibody with the expected heavy and light chain protein masses. [Figure 4] (A-B) are a series of line graphs showing antigen binding of light chain-paired bispecific antibodies shown in Figures 2A-2B compared to isotype and bispecific antibody controls. (A) is a line graph showing antigen binding of light chain-paired bispecific antibody variants by sandwich ELISA with antibody capture on CD3ε coated plates. (B) is a line graph showing antigen binding of light chain-paired bispecific antibody variants by sandwich ELISA with antibody capture on recombinant ULBP2 coated plates. [Figure 5A] Mass spectrometry analysis of EIP0205 showing the intact mass after PNGase F deglycosylation under non-reducing conditions and chromatographic separation using a reversed-phase C4 column. [Figure 5B] Mass spectrometry analysis of EIP0205 showing the reduced mass of the heavy chain after rapid PNGase F deglycosylation under reducing conditions and chromatographic separation using a reversed phase C4 column. [Figure 5C] Mass spectrometry analysis of EIP0205 showing the reduced mass of the light chain after rapid PNGase F deglycosylation under reducing conditions and chromatographic separation using a reversed-phase C4 column. [Figure 5D] Mass spectrometry analysis of EIP0187 showing the intact mass after PNGase F deglycosylation under non-reducing conditions and chromatographic separation using a reversed-phase C4 column. [Figure 5E] Mass spectrometry analysis of EIP0187 showing the reduced mass of the heavy chain after rapid PNGase F deglycosylation under reducing conditions and chromatographic separation using a reversed phase C4 column. [Figure 5F]Mass spectrometry analysis of EIP0187 showing the reduced mass of the light chain after rapid PNGase F deglycosylation under reducing conditions and chromatographic separation using a reversed-phase C4 column. [Figure 6] FIG. 2C is a line graph showing functional evaluation (cytotoxicity) of light chain paired bispecific antibodies shown in FIGS. 2A-2B in a tumor cell and T cell co-culture assay compared to a bispecific control antibody (EIP0112). [Figure 7A] Chromatograms of bispecific antibody variants obtained from tandem purification. [Figure 7B] Non-reducing NuPAGE analysis showing protein mass of intact bispecific antibody variants. [Figure 7C] FIG. 13 is a reducing NuPAGE analysis showing the protein masses of the heavy and light chains of bispecific antibody variants. [Figure 7D] FIG. 1 is a line graph showing antigen binding of light chain-paired bispecific antibodies by sandwich ELISA, with antibody captured on CD3ε coated plates. [Figure 7E] FIG. 1 is a line graph showing antigen binding of light chain-paired bispecific antibodies by sandwich ELISA, with antibody captured on antibody-coated plates. [Figure 8A] 1 is a line graph showing binding of αULBP2-αCD3 bispecific antibody variants to human CD3 epsilon by ELISA. [Figure 8B] 1 is a line graph showing binding of αULBP2-αCD3 bispecific variants to cynomolgus CD3 epsilon by ELISA. [Figure 9A] 1 is a line graph showing luciferase activity in co-cultures of tumor cells and Jurkat NFAT luciferase reporter cells in the presence of αULBP2-αCD3 bispecific antibody variants. [Figure 9B]1 is a line graph showing luciferase activity in co-cultures of tumor cells and Jurkat NFAT luciferase reporter cells in the presence of αULBP2-αCD3 bispecific antibody variants. [Figure 10A] 9A-9B are line graphs showing T cell mediated cytotoxicity of tumor cell lines in the presence of the αULBP2-αCD3 bispecific antibody variants of Figures 9A-9B. 9B shows cytotoxicity of HCT116 tumor cells. [Figure 10B] Figure 9 is a line graph showing T cell mediated cytotoxicity of tumor cell lines in the presence of the αULBP2-αCD3 bispecific antibody variants of Figures 9A-9B. Figure 9 shows cytotoxicity of MDA-MB-231 GFP tumor cells. [Figure 10C] Figure 9 is a line graph showing T cell mediated cytotoxicity of tumor cell lines in the presence of the αULBP2-αCD3 bispecific antibody variants of Figures 9A-9B.Figure 9 shows cytotoxicity of SiHa tumor cells in the presence of the αULBP2-αCD3 bispecific antibody variants. [Figure 11A] 9A-9B are line graphs showing cytokine secretion from activated T cells in co-culture with SiHa tumor cells in the presence of the αULBP2-αCD3 bispecific affinity variants of Figures 9A-9B. IFNγ secretion is shown. [Figure 11B] 9A-9B are line graphs showing cytokine secretion from activated T cells in co-culture with SiHa tumor cells in the presence of the αULBP2-αCD3 bispecific affinity variants of Figures 9A-9B. IL-2 secretion is shown. [Figure 11C] 9A-9B are line graphs showing cytokine secretion from activated T cells in co-culture with SiHa tumor cells in the presence of the αULBP2-αCD3 bispecific affinity variants of Figures 9A-9B. [Figure 12A] 1 shows bispecific antibody variants without the CD58 fusion. [Figure 12B] 1 shows a bispecific antibody variant with a CD58 fusion to the carboxyl terminus of CH1H3. [Figure 12C]FIG. 1 shows a bispecific antibody variant with a CD58 fusion to the carboxyl terminus of CH2H3. [Figure 12D] FIG. 1 shows bispecific antibody variants with a CD58 fusion to the carboxyl terminus of CH1H3 and a CD58 fusion to the carboxyl terminus of CH2H3. [Figure 12E] 1 shows a bispecific antibody variant with an amino-terminal fusion to CD58 in VL2. [Figure 12F] 1 shows a bispecific antibody variant with an amino-terminal fusion to CD58 in VH2. [Figure 12G] 1 shows a bispecific antibody variant with an amino-terminal fusion to CD58 in VL1. [Figure 12H] 1 shows a bispecific antibody variant with an amino-terminal fusion to CD58 in VH1. [Figure 12L] 1 shows a bispecific antibody variant with a CD58 fusion to the carboxyl terminus of CL2. [Figure 12J] 1 shows a bispecific antibody variant with a CD58 fusion to the carboxyl terminus of CL1. [Figure 12K] 1 shows bispecific antibody variants with CD58 fusions to the carboxyl termini of CL1 and CL2. [Figure 13] Chromatograms obtained from tandem purification of co-stimulatory ligand or cytokine fusion bispecific variants (EIP0205, EIP0359, EIP0360, EIP0363) engineered using light chain pairing technology. [Figure 14] Differential scanning calorimetry analysis of costimulatory ligand or cytokine fusion αULBP2-αCD3 bispecific variants (EIP0205, EIP0359, EIP0363). [Figure 15A] FIG. 1 is a line graph showing antigen binding of costimulatory ligand or cytokine fused bispecific antibody variants to recombinant CD3ε coated plates by sandwich ELISA. [Figure 15B]FIG. 1 is a line graph showing antigen binding of costimulatory ligand or cytokine fused bispecific antibody variants to plates coated with recombinant ULBP2 protein by sandwich ELISA. [Figure 16A] Shows cytolysis of MDA-MB-231 GFP tumor cells in the presence of αULBP2-αCD3 bispecific variants after 7 days of incubation with naive T cells. [Figure 16B] Representative bright field and fluorescence microscopy images of naive T cell activation and MDA-MB-231 cell death are shown. [Figure 16C] Shows cytolysis of ULBP2-deficient MDA-MB-231 GFP tumor cells in the presence of αULBP2-αCD3 bispecific antibody variants after incubation with naive T cells for 7 days. [Figure 17A] FIG. 16C is a line graph showing IFNγ secretion from naive T cells after 24 hours of incubation with MDA-MB-231 GFP tumor cells and ULBP2-deficient MDA-MB-231 GFP tumor cells in the presence of the bispecific antibody variants of FIGS. 16A-16C. MDA-MB-231 GFP tumor cells are shown. [Figure 17B] 16A-16C are line graphs showing IFNγ secretion from naive T cells after 24 hours of incubation with MDA-MB-231 GFP tumor cells and ULBP2-deficient MDA-MB-231 GFP tumor cells in the presence of the bispecific antibody variants of Figures 16A-16C. ULBP2-deficient MDA-MB-231 GFP tumor cells are shown. [Figure 18A] FIG. 16C is a line graph showing IL-2 secretion from naive T cells after 24 hours of incubation with MDA-MB-231 GFP tumor cells and ULBP2-deficient MDA-MB-231 GFP tumor cells in the presence of the bispecific antibody variants of FIGS. 16A-16C. MDA-MB-231 GFP tumor cells are shown. [Figure 18B]16A-16C are line graphs showing IL-2 secretion from naive T cells after 24 hours of incubation with MDA-MB-231 GFP tumor cells and ULBP2-deficient MDA-MB-231 GFP tumor cells in the presence of the bispecific antibody variants of Figures 16A-16C. ULBP2-deficient MDA-MB-231 GFP tumor cells are shown. [Figure 19A] FIG. 16C is a line graph showing IFNγ secretion from naive T cells after 24 hours of incubation with MDA-MB-231 GFP tumor cells and ULBP2-deficient MDA-MB-231 GFP tumor cells in the presence of the bispecific antibody variants of FIGS. 16A-16C. MDA-MB-231 GFP tumor cells are shown. [Figure 19B] 16A-16C are line graphs showing IFNγ secretion from naive T cells after 24 hours of incubation with MDA-MB-231 GFP tumor cells and ULBP2-deficient MDA-MB-231 GFP tumor cells in the presence of the bispecific antibody variants of Figures 16A-16C. ULBP2-deficient MDA-MB-231 GFP tumor cells are shown. [Figure 20A] Cytolysis of SiHa cells in the presence of αULBP2-αCD3 bispecific variant and αULBP2-αCD3-CD58 bispecific variant after 48 hours of incubation with activated T cells. [Figure 20B] Cytolysis of SiHa cells in the presence of αULBP2-αCD3 bispecific variant and αULBP2-αCD3-CD58 bispecific variant after 48 hours of incubation with activated T cells. [Figure 21A] Cytolysis of SiHa cells in the presence of αULBP2-αCD3 bispecific variant and αULBP2-αCD3-CD58 bispecific variant after 48 hours of incubation with activated T cells. [Figure 21B] Cytolysis of SiHa cells in the presence of αULBP2-αCD3 bispecific variant and αULBP2-αCD3-CD58 bispecific variant after 48 hours of incubation with activated T cells. [Figure 22A]FIG. 1 is a line graph showing IFNγ secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 22B] FIG. 1 is a line graph showing IFNγ secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 23A] FIG. 1 is a line graph showing IFNγ secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 23B] FIG. 1 is a line graph showing IFNγ secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 24A] FIG. 1 is a line graph showing IL-2 secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 24B] FIG. 1 is a line graph showing IL-2 secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 25A] FIG. 1 is a line graph showing IL-2 secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 25B]FIG. 1 is a line graph showing IL-2 secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 26A] FIG. 1 is a line graph showing TNFα secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 26B] FIG. 1 is a line graph showing TNFα secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 27A] FIG. 1 is a line graph showing TNFα secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 27B] FIG. 1 is a line graph showing TNFα secretion after 48 hours of activated T cells in co-culture with SiHa cells in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants. [Figure 28] FIG. 13 is a line graph showing cytolysis of MDA-MB-231 GFP cells after 7 days of incubation with PBMCs in the presence of αULBP2-αCD3, αULBP2-αCD3-CD58 bispecific αULBP2-αCD3-CD58 variable domain fusion bispecific antibody variants. [Figure 29A] FIG. 1 is a line graph showing cytolysis of MDA-MB-231 cells in the presence of bispecific antibody variants. [Figure 29B] FIG. 1 is a line graph showing IFNγ secretion from MDA-MB-231 cells in the presence of bispecific antibody variants. [Diagram 30]Representative microscopy images of cytolysis of MDA-MB-231-GFP cells after 48 h of incubation with naive, round 3 and round 5 exhausted T cells in the presence of bispecific antibody variants. [Diagram 31] FIG. 11 is a radar plot showing normalized levels of T cell markers IL2, IFNγ, CD25, CD69, GZMB, CD2, PD-1, CD38 and TIM3 present after 72 hours of incubation of PBMCs with MDA-MB-231 cells in the presence of bispecific antibody variants. [Figure 32A] 1 is a graph showing improved tumor growth inhibition in humanized mice following treatment with bispecific antibody variants. FIG. 2 is a line graph showing growth inhibition of SiHa tumors over time following adoptive transfer of human T cells and dosing with bispecific antibody variants EIP0542, EIP0205 and EIP0359. [Figure 32B] 32A-32C are graphs showing improved survival in humanized mice following treatment with bispecific antibody variants.Survival curves following adoptive transfer of human T cells and dosing with the bispecific antibody variants of FIG. [Figure 32C] Figure 32A is a graph showing improved pharmacokinetics in humanized mice following treatment with bispecific antibody variants.Figure 32B is a line graph showing the pharmacokinetics of the bispecific antibody variants of Figure 32A. [Fig. 32D] Figure 1 shows improved pharmacokinetics in humanized mice following treatment with bispecific antibody variants.Figure 2 shows the pharmacokinetics of EIP0561 following administration of a 10 mg / kg dose to humanized mice. [Diagram 33] (A-C) are a series of histograms from flow cytometry analysis of tumor infiltrating lymphocytes in SiHa tumors 3 days after engraftment and treated with bispecific antibody variants. (A) Granzyme B analysis showing increased tumor cell lysis with bispecific antibody variants. (B) CD25 analysis showing increased T cell activation with bispecific antibody variants. (C) CD38 analysis showing decreased T cell exhaustion with bispecific antibody variants. [Figure 34A]Structural rendering modeling the binding of A06 and E12 to ULBP2. Homology model of the ULBP2-NKG2D complex. Residue R106 is shown in stick model. ULBP2 is shown in dark grey, NKG2D, A06, and E12 are shown in light grey. [Figure 34B] Structural rendering modeling the binding of A06 and E12 to ULBP2. Docking model of ULBP2 and the A06 antibody. Residue R106 is shown in stick model. ULBP2 is shown in dark grey, NKG2D, A06, and E12 are shown in light grey. [Figure 34C] Structural rendering modeling the binding of A06 and E12 to ULBP2. Docking model of ULBP2 and E12 antibody. Residue R106 is shown in stick model. ULBP2 is shown in dark grey, NKG2D, A06, and E12 are shown in light grey. [Diagram 35] FIG. 1 is a line graph showing growth inhibition of CORL-105 tumors over time following co-engraftment of human T cells and dosing of bispecifics with various CD3 affinities and bispecific CD58 fusions. [Figure 36A] Figure 1 is a graph showing cytolysis of tumor cells in the presence of bispecific CD58 fusions after 48 hours of incubation with activated T cells.Figure 1 shows cytolysis of HCT116 cells. [Figure 36B] Figure 1 is a graph showing cytolysis of tumor cells in the presence of bispecific CD58 fusions after 48 hours of incubation with activated T cells.Figure 1 shows cytolysis of U266B1 cells. [Figure 36C] Figure 1 is a graph showing cytolysis of tumor cells in the presence of bispecific CD58 fusions after 48 hours of incubation with activated T cells.Figure 1 shows cytolysis of JeKo-1 cells. [Figure 36D] 1 is a graph showing cytolysis of tumor cells in the presence of bispecific CD58 fusions after 48 hours of incubation with activated T cells.Cytolysis of PSMA-low LNCAP prostate cancer cells (LNCAP-vL) is shown. [Figure 36E] Figure 1 is a graph showing cytolysis of tumor cells in the presence of bispecific CD58 fusions after 48 hours of incubation with activated T cells. [Figure 36F] Figure 1 is a graph showing cytolysis of tumor cells in the presence of bispecific CD58 fusions after 48 hours of incubation with activated T cells.Figure 2 shows cytolysis of Raji cells. [Figure 37] 1 is a graph depicting chromatograms obtained from tandem purification of an exemplary antibody with and without an exemplary disulfide stabilizing mutation. [Figure 38A] 1 is a graph showing cytolysis of MDA-MB-231 GFP and ULBP2-deficient MDA-MB-231 GFP tumor cells in the presence of αULBP2-αCD3 bispecific antibody variants after 5 days of incubation with naive T cells at a 1:10 ratio.Tumor cells incubated with various concentrations of EIP0205 are shown. [Figure 38B] 13A-C are microscopy images showing cytolysis of MDA-MB-231 GFP and ULBP2-deficient MDA-MB-231 GFP tumor cells in the presence of αULBP2-αCD3 bispecific antibody variants after 5 days of incubation with naive T cells at a ratio of 1:10. Representative bright field and fluorescence microscopy images of naive T cell activation and MDA-MB-231 cell death by EIP0205 are shown. [Figure 38C] 1 is a graph showing cytolysis of MDA-MB-231 GFP and ULBP2-deficient MDA-MB-231 GFP tumor cells in the presence of αULBP2-αCD3 bispecific antibody variants after 5 days of incubation with naive T cells at a 1:10 ratio.Tumor cells incubated with various concentrations of EIP0359 are shown. [Figure 38D]13A-C are microscopy images showing cytolysis of MDA-MB-231 GFP and ULBP2-deficient MDA-MB-231 GFP tumor cells in the presence of αULBP2-αCD3 bispecific antibody variants after 5 days of incubation with naive T cells at a ratio of 1:10. Representative bright field and fluorescence microscopy images of naive T cell activation and MDA-MB-231 cell death by EIP0359 are shown. [Figure 39] FIG. 13 is a graph showing cytolysis of MDA-MB-231 GFP tumor cells in the presence of αULBP2-αCD3 bispecific antibody variants after incubation with naive T cells at a ratio of 1:10 for 5 days. [Figure 40A] 1 is a line graph showing tumor cell cytolysis in the presence of αULBP2-αCD3 bispecific and αULBP2-αCD3-CD58 bispecific variants after incubation with naive T cells at an E:T ratio of 10:1 for 5 days. [Figure 40B] 1 is a line graph showing cytokine secretion in the presence of αULBP2-αCD3 bispecific and αULBP2-αCD3-CD58 bispecific variants after 5 days of incubation with naive T cells at an E:T ratio of 10:1. IFNγ secretion after 48 hours of activated T cells in co-culture with tumor cells is shown. [Figure 40C] 1 is a line graph showing cytokine secretion in the presence of αULBP2-αCD3 bispecific and αULBP2-αCD3-CD58 bispecific variants after 5 days of incubation with naive T cells at an E:T ratio of 10:1. IL-2 secretion after 48 hours of activated T cells in co-culture with tumor cells is shown. [Diagram 41] FIG. 13 is a graph showing killing of MDA-MB-231 tumor cells in the presence of activated T cells in the presence of αULBP2-αCD3-CD58 bispecific variant after 48 hours of incubation with activated T cells at an E:T ratio of 5:1. [Figure 42A]Graph showing tumor cell cytolysis in the presence of activated T cells in the presence of αCD3 bispecific antibody variants with light chain pairing of the present disclosure after 2 days compared to control.JeKo-1 tumor cells are shown at an effector:target cell (E:T) ratio of 5:1. [Figure 42B] Graph showing tumor cell lysis in the presence of activated T cells in the presence of αCD3 bispecific antibody variants with light chain pairing of the present disclosure after 2 days compared to control Ramos cells at an effector:target cell (E:T) ratio of 10:1. [Figure 42C] Graph showing tumor cell lysis in the presence of activated T cells in the presence of αCD3 bispecific antibody variants with light chain pairing of the present disclosure after 2 days compared to control.Raji cells are shown at an effector:target cell (E:T) ratio of 10:1. [Fig.42D] Graph showing tumor cell cytolysis in the presence of activated T cells in the presence of αCD3 bispecific antibody variants with light chain pairing of the present disclosure after 2 days compared to control SUDHL10 cells at an effector:target cell (E:T) ratio of 10:1. [Figure 42E] Graph showing tumor cell cytolysis in the presence of activated T cells in the presence of αCD3 bispecific antibody variants with light chain pairing of the present disclosure after 2 days compared to control MV411 cells at an effector:target cell (E:T) ratio of 5:1. [Fig.42F] Graph showing tumor cell cytolysis in the presence of activated T cells in the presence of αCD3 bispecific antibody variants with light chain pairing of the present disclosure after 2 days compared to control OCI-AML2 cells at an effector:target cell (E:T) ratio of 5:1. [Figure 43A] Graph showing tumor cell lysis in the presence of naive T cells at an effector:target cell (E:T) ratio of 7.5:1 in the presence of αCD3 bispecific antibody variants comprising light chain pairing with and without a CD58 fusion molecule of the present disclosure after 3 days compared to a control. [Figure 43B]1 is a graph showing tumor cell lysis in the presence of naive T cells at an effector:target cell (E:T) ratio of 7.5:1 in the presence of αCD3 bispecific antibody variants comprising light chain pairing with and without a CD58 fusion molecule of the present disclosure after 3 days compared to a control. [Diagram 44] (A-B) are a series of graphs showing cytolysis of tumor cells in the presence of activated T cells in the presence of αBCMA-αCD3 and αBCMA-αCD3-CD58 bispecific variants after 48 hours of incubation compared to a no antibody control. Figure 43A shows NCI929 tumor cells at an effector:target cell (E:T) ratio of 2:1. Figure 43B shows U266B1 cells at an effector:target cell (E:T) ratio of 1.6:1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] The production of heteromultimeric antibodies (e.g., bispecific antibodies) using conventional techniques brings challenges, including, among others, the production of mispaired antibodies, reduced yields, and reduced / lost effector functions. Furthermore, aggregation and precipitation often occur during the preparation and assembly of heteromultimeric antibodies. Mismatches between heavy and light chains, aggregation and precipitation can greatly reduce the yield of desired heteromultimeric antibodies. Thus, there is a need for compositions and methods herein for more efficient and high-level production of heteromultimeric antibodies.

[0068] Disclosed herein are compositions and efficient production processes / methods for economically producing heteromultimeric antibodies (e.g., bispecific antibodies) by using or adjusting one or more of the following, including but not limited to, oppositely charged mutation pairs in the light and heavy chains, disulfide bond rearrangement mutations, or knobs-in-holes mutations. The inventive methods described herein reduce mismatches of heavy and light chains, reduce protein loss due to precipitation and / or aggregation, and improve the overall yield of heteromultimeric antibody production, including bispecific antibody production.

[0069] T cell retargeting (or T cell redirecting) bispecific antibodies are a novel class of therapeutic agents that can recruit T cells to tumor cells and induce tumor-specific (but MHC-independent) activation of T cell effector activity. Typically, T cell retargeting bispecific antibodies contain an antigen-binding domain that targets the CD3 portion of the T cell receptor for T cell recruitment and an antigen-binding domain that targets a disease-associated antigen (DAA). This targeting design promotes the recruitment of T cells and positions them closely with the target tumor cells, forming an immunological synapse and activating local T cells, which then destroy the target cells by perforin and granzymes released from T cell cytotoxic granules.

[0070] Since the CD3 binding affinity of T cell retargeting bispecific antibodies is essential for T cell recruitment, the present invention also relates to the generation of a panel of antibodies that bind human CD3 and cross-react with cynomolgus CD3. Cross-reactivity with cynomolgus CD3 is an important feature to facilitate preclinical development of T cell retargeting bispecific antibodies incorporating the anti-CD3ε antibodies described herein. Furthermore, these anti-CD3ε antibodies exhibit a range of binding affinities. The affinity of the CD3 arms of a bispecific antibody can significantly modify the functional activity of the bispecific antibody. It is therefore desirable and advantageous to have anti-CD3 antibodies with a range of affinities.

[0071] Additionally, the bispecific antibodies disclosed herein may have cytokine or costimulatory molecule fusion peptides that act as antagonists to inhibit or block deleterious interactions, or as agonists to mimic or enhance physiological responses, including but not limited to T cell activation, T cell proliferation, and prevention of T cell exhaustion. These properties are advantageous over conventional CD3 bispecific antibodies or tumor-targeted costimulatory receptor agonists that do not optimally activate T cells and induce (or promote) T cell dysfunction. Thus, cytokines and / or costimulatory fusion peptides are advantageous in enhancing the therapeutic potential of bispecific antibodies.

[0072] Overall, these features (e.g., mutation pairs with opposite charges in the light and heavy chains, disulfide bond rearrangement mutations, knob-into-hole mutations, CD3 affinity variants, cytokine or costimulatory molecule fusion peptides) present considerable advantages from a development perspective. For example, it results in T cell costimulation and prevents T cell exhaustion. Moreover, bispecific T cell retargeting agents share the drug-like properties of human monoclonal antibodies. Moreover, T cell retargeting bispecific antibodies are advantageous over other existing therapies (e.g., CAR-T therapy) because they offer an off-the-shelf product with a high safety profile (e.g., mitigation of cytokine release syndrome and reduced levels of tonic signaling leading to T cell dysfunction) as well as the possibility of dose titration and titration.

[0073] Antibody composition and structure

[0074] The present disclosure provides an antibody having the following domain structure: a) a variable region (VH1), and a constant region 1 domain (CH1 H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domains (CH1 H3 a) a first heavy chain polypeptide (H1) comprising a constant region (CH1) having a variable region (VL1) and a constant region (CL1); and b) a first light chain polypeptide (L1) comprising a variable region (VH2), and a constant region 1 domain (CH2 H1 ), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3 The present disclosure provides an antibody comprising a second heavy chain polypeptide (H2) comprising a constant region (CH2) having a variable region (VL2) and a constant region (CL2). Schematic diagrams of the antibody structures of the present disclosure are shown in Figures 1A-1D.

[0075] As used herein, the term "antibody" refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules (i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen). "Specifically binds to" or "immunoreacts with" or "directed against" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides, or does so with a significantly lower affinity (K d >10 -6 ) means to bind to the antibody. Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, and chimeric antibodies. Antibodies may be obtained from recombinant sources and / or produced in transgenic animals.

[0076] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0077] Generally, antibody molecules obtained from humans are associated with any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other depending on the nature of the heavy chain present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, IgG4, etc. Furthermore, in humans, the light chains can be kappa or lambda chains. Thus, in one embodiment, the antibodies disclosed herein are IgG antibodies.

[0078] Antibodies can be purified by well-known techniques such as affinity chromatography using Protein A or Protein G, which yields primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen or epitope that is the target of the desired immunoglobulin may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is described, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0079] The term "antibody fragment" as used herein is intended to include, but is not limited to, Fv, Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies and multimers thereof, multispecific antibody fragments, and domain antibodies. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating antibodies with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques.

[0080] Techniques can be adapted to produce single chain antibodies specific to antigenic proteins of the disclosure (see, e.g., U.S. Patent No. 4,946,778). Moreover, methods can be adapted for the construction of Fab expression libraries which allow rapid and efficient identification of monoclonal Fab fragments with the desired specificity for a protein or a derivative, fragment, analog or homolog thereof (see, e.g., Huse, et al., 1989 Science 246:1275-1281).

[0081] As used herein, the term "epitope" refers to a site on an antigen that is recognized by the antibodies and fragments disclosed herein. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin. Epitope determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics and specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is less than 1 micromolar, e.g., less than 100 nM, preferably less than 10 nM, more preferably less than 1 nM.

[0082] A bispecific antibody is an antibody that has binding specificities for at least two different antigens. The present disclosure provides a bispecific antibody having a first antigen-binding region that binds to a first antigen (e.g., CD3) and a second antigen-binding region that binds to a second antigen (e.g., a disease-associated antigen).

[0083] Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0084] Antibody variants

[0085] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the heavy chain heterodimerization, light chain heterodimerization, binding affinity, and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion of residues from, and / or insertion of residues into, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct has the desired properties (e.g., light chain heterodimerization, heavy chain heterodimerization, antigen binding).

[0086] Amino acids can be classified according to the following general side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic (negatively charged): Asp, Glu, (4) Basic (positively charged): His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0087] Functional variants of the antibodies or antigen-binding fragments described herein are also encompassed by the present disclosure. The term "functional variant" as used herein includes modifications or chemical equivalents of the amino acid and nucleic acid sequences disclosed herein that perform substantially the same function as the polypeptides or nucleic acid molecules disclosed herein. For example, functional variants of the polypeptides disclosed herein include, but are not limited to, conservative amino acid substitutions.

[0088] As used herein, a "conservative amino acid substitution" is one in which one amino acid residue is replaced with another, thereby changing the amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). Variants of polypeptides also include additions and deletions to the polypeptide sequences disclosed herein. In addition, variant nucleotide sequences include analogs and derivatives thereof. Variants of the binding proteins disclosed herein include proteins that bind to the same antigen or epitope as the binding proteins.

[0089] In some embodiments, the charged amino acid residue is a naturally occurring or non-naturally occurring amino acid, hi some embodiments, the naturally occurring charged amino acid residue is arginine, lysine, histidine, glutamic acid, or aspartic acid.

[0090] Light and Heavy Chain Substitution Variants

[0091] To generate a substantially homogenous population of bispecific antibodies with proper pairing of heavy and light chains (i.e., the cognate pairing or heterodimerization of light and heavy chains necessary to form the original antibody variable domain or antigen-binding domain), the first heavy chain polypeptide (H1) has a strong preference for binding to the first light chain polypeptide (L1) relative to the second light chain polypeptide (L2), and the second heavy chain polypeptide (H2) has a strong preference for binding to the second light chain polypeptide (L2) relative to the first light chain polypeptide (L1). Furthermore, the first heavy chain polypeptide (H1) and the second heavy chain polypeptide (H2) have a strong preference for heterodimerization over homodimerization (i.e., heavy chain heterodimerization).

[0092] Provided herein are antibody variants having one or more amino acid substitutions. Exemplary substitution mutagenesis sites include the charge substitution pairs shown in Tables 1.1-1.3 and Tables 2-6.

[0093] In the bispecific antibodies of the present disclosure, it is advantageous to use a Fab heterodimerization strategy that allows for proper association of Fab domains belonging to the same arm and minimizes aberrant pairing of Fab domains belonging to different arms. Exemplary Fab heterodimerization strategies include, but are not limited to, those shown in Tables 1.1 and 1.2. The antibody domains listed in Tables 1.1 and 1.2 correspond to the domains of the present disclosure shown in Figure 1.

[0094] [Table 1]

[0095] [Table 2-1] [Table 2-2]

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

[0096]

Table 3

[0097]

Table 4

[0098]

Table 5

[0099]

Table 6

[0100]

Table 7

[0101]

Table 8

[0102] In certain embodiments, the antibody variant comprises the following substitutions: i) the amino acid at position 39 (Kabat numbering) of VH1 and VH2 is a charged or polar amino acid residue and the amino acid at position 38 (Kabat numbering) of VL1 and VL2 is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 39 of VH1 and VH2, or the amino acid at position 100 (Kabat numbering) of VH1 and VH2 is a charged or polar amino acid residue and the amino acid at position 44 (Kabat numbering) of VL1 and VL2 is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 100 of VH1 and VH2; ii) the CH1 H1 and CH1 H2 The amino acid at position 147 (EU numbering) of CL1 or CL2 is a charged or polar amino acid residue, and one of the amino acids at positions 131, 179 or 180 (EU numbering) of CL1 or CL2 is a CH1 H1 and CH1 H2 and iii) an amino acid residue having an opposite charge or polarity compared to the amino acid at position 147 of CH1. H1 and CH1 H2 The amino acid at position 185 (EU numbering) of CL1 is a charged or polar amino acid residue, and the amino acid at position 137 (EU numbering) of CL1 and CL2 is a CH1 H1 and CH1 H2 or an amino acid residue having an opposite charge or polarity compared to the amino acid at position 185 of CH1 H1 and CH1 H2 The amino acid at position 187 (EU numbering) of CL1 is a charged or polar amino acid residue, and one of the amino acids at positions 137 or 138 (EU numbering) of CL1 and CL2 is a CH1 H1 and CH1 H2 and iv) an amino acid residue having an opposite charge or polarity compared to the amino acid at position 187 (EU numbering) of CH1. H1 and CH1 H2The amino acid at position 145 (EU numbering) of CL1 is a charged or polar amino acid residue, and the amino acid at position 131 (EU numbering) of CL1 and CL2 is a CH1 H1 and CH1 H2 It is an amino acid residue that has an opposite charge or polarity compared to the amino acid at position 145 of.

[0103] In certain embodiments, the antibody variant comprises the following substitutions: the H1 amino acids at positions 39, 100, 147, 185, 187 or 145 are positively charged, the L1 amino acids at positions 38, 44, 131, 179, 180, 137 or 138 are negatively charged, the H2 amino acids at positions 39, 100, 147, 185, 187 or 145 are negatively charged, and the L2 amino acids at positions 38, 44, 131, 179, 180, 137 or 138 are positively charged.

[0104] In certain embodiments, the antibody variant comprises the following substitutions: the H1 amino acids at positions 39, 100, 147, 185, 187 and 145 are negatively charged, the L1 amino acids at positions 38, 44, 131, 179, 180, 137 or 138 are positively charged, the H2 amino acids at positions 39, 100, 147, 185, 187 or 145 are positively charged, and the L2 amino acids at positions 38, 44, 131, 179, 180, 137 or 138 are negatively charged.

[0105] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set A" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 and the amino acid at position 137 (EU numbering) of CL1 is D; H1and iv) the amino acid at position 220 (EU numbering) in H1H is S and the amino acid at position 214 (EU numbering) in CL1 is S; b) H2 and L2 include: i) VH2 where the amino acid at position 39 (Kabat numbering) is D and the amino acid at position 38 (Kabat numbering) in VL2 where K; and ii) CH2 where the amino acid at position 220 (EU numbering) in H1H is S and the amino acid at position 214 (EU numbering) in CL1 is S. H1 and the amino acid at position 180 (EU numbering) of CL2 is R.

[0106] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set B" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1 and CL1 has an amino acid at position 137 (EU numbering) that is D; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is D and VL2 has an amino acid at position 38 (Kabat numbering) that is K; ii) CH2 H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0107] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set C" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1and CL1 has an amino acid at position 137 (EU numbering) that is D; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is D and VL2 has an amino acid at position 38 (Kabat numbering) that is K; ii) CH2 H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0108] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set D" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 and the amino acid at position 131 (EU numbering) of CL1 is D; iii) CH1 H1 and iv) the amino acid at position 220 (EU numbering) in H1H is S and the amino acid at position 214 (EU numbering) in CL1 is S; b) H2 and L2 include: i) VH2 where the amino acid at position 39 (Kabat numbering) is D and the amino acid at position 38 (Kabat numbering) in VL2 is K; and ii) CH2 where the amino acid at position 220 (EU numbering) in H1H is S and the amino acid at position 214 (EU numbering) in CL1 is S. H1 and the amino acid at position 180 (EU numbering) of CL2 is R.

[0109] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set E" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 and the amino acid at position 137 (EU numbering) of CL1 is D; H1 and iv) the amino acid at position 220 (EU numbering) in H1H is S and the amino acid at position 214 (EU numbering) in CL1 is S; b) H2 and L2 include: i) the amino acid at position 39 (Kabat numbering) in VH2 is D and the amino acid at position 38 (Kabat numbering) in VL2 is K; and ii) CH2 H1 and the amino acid at position 180 (EU numbering) of CL2 is R.

[0110] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set F" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1 in which the amino acid at position 185 (EU numbering) is K and in CL1 at position 137 (EU numbering) is D; b) H2 and L2 include: i) VH2 in which the amino acid at position 39 (Kabat numbering) is D and in VL2 in which the amino acid at position 38 (Kabat numbering) is K; ii) CH2 H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0111] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set G" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1 and CL1 has an amino acid at position 137 (EU numbering) that is D; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is D and VL2 has an amino acid at position 38 (Kabat numbering) that is K; ii) CH2 H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0112] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set H" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1and iii) CL1 having an amino acid at position 179 (EU numbering) of E; b) H2 and L2 include: i) VH2 having an amino acid at position 39 (Kabat numbering) of D and VL2 having an amino acid at position 38 (Kabat numbering) of K; ii) CH2 having an amino acid at position 185 (EU numbering) of E and CL1 having an amino acid at position 137 (EU numbering) of K; and iii) CL1 having an amino acid at position 179 (EU numbering) of E; H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0113] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set I" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1 and CL1 has an amino acid at position 137 (EU numbering) that is D; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is D and VL2 has an amino acid at position 38 (Kabat numbering) that is K; ii) CH2 H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0114] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set J" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 and iii) CL1 having an amino acid at position 179 (EU numbering) of E; b) H2 and L2 include: i) VH2 having an amino acid at position 39 (Kabat numbering) of D and VL2 having an amino acid at position 38 (Kabat numbering) of K; ii) CH2 having an amino acid at position 185 (EU numbering) of E and CL1 having an amino acid at position 137 (EU numbering) of K; and iii) CL1 having an amino acid at position 179 (EU numbering) of E; H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0115] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set K" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1 and CL1 has an amino acid at position 137 (EU numbering) that is D; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is D and VL2 has an amino acid at position 38 (Kabat numbering) that is K; ii) CH2 H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0116] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set L" that includes the following substitutions: a) H1 and L1 are: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 and the amino acid at position 131 (EU numbering) of CL1 is D; iii) CH1 H1 and CL1 has an amino acid at position 137 (EU numbering) that is D; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is D and VL2 has an amino acid at position 38 (Kabat numbering) that is K; ii) CH2 H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1 and the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0117] In certain embodiments, the antibody variant comprises "Light Chain Pairing Mutation Set 0340" comprising the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is D and the amino acid at position 38 (Kabat numbering) of VL1 is K; and ii) CH1 H1and CL1 has an amino acid at position 137 (EU numbering) that is K; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is K and VL2 has an amino acid at position 38 (Kabat numbering) that is D; ii) CH2 H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0118] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set M" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is D and the amino acid at position 38 (Kabat numbering) of VL1 is K; and ii) CH1 H1 and CL1 has an amino acid at position 137 (EU numbering) that is K; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is K and VL2 has an amino acid at position 38 (Kabat numbering) that is D; ii) CH2 H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0119] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set N" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is D and the amino acid at position 38 (Kabat numbering) of VL1 is K; and ii) CH1 H1 and CL1 having an amino acid at position 137 (EU numbering) that is D; b) H2 and L2 include: i) VH2 having an amino acid at position 39 (Kabat numbering) that is K and VL2 having an amino acid at position 38 (Kabat numbering) that is D; ii) CH2 H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0120] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set O" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is D and the amino acid at position 38 (Kabat numbering) of VL1 is K; ii) CH1 H1 and the amino acid at position 131 (EU numbering) of CL1 is D; iii) CH1 H1 and iv) CH1 H1 and CL1 has an amino acid at position 180 (EU numbering) that is E; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is K and VL2 has an amino acid at position 38 (Kabat numbering) that is D; ii) CH2 H1and the amino acid at position 138 (EU numbering) of CL2 is K; iii) the amino acid at position 170 (EU numbering) of VH2 is C and the amino acid at position 162 (EU numbering) of VL2 is C; and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) of CL2 is S.

[0121] In certain embodiments, the antibody variant comprises "Light Chain Pairing Mutation Set 367" comprising the following substitutions: a) H1 and L1 are: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 and iii) CH1 H1 and CL1, wherein the amino acid at position 185 (EU numbering) is E and the amino acid at position 137 (EU numbering) of CL1 is D; b) H2 and L2 include: i) VH2, wherein the amino acid at position 39 (Kabat numbering) is D and VL2, wherein the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2, wherein the amino acid at position 185 (EU numbering) of CL1 is E and the amino acid at position 137 (EU numbering) of CL1 is D; H1 and the amino acid at position 137 (EU numbering) of CL2 is K; iii) the amino acid at position 138 (EU numbering) of CL2 is R; iv) CH2 H1 and v) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0122] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set P" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii) CH1H1 and iii) CH1 H1 and b) H2 and L2 are as follows: i) VH2 has an amino acid at position 39 (Kabat numbering) that is D and VL2 has an amino acid at position 38 (Kabat numbering) that is K; ii) CH2 has an amino acid at position 145 (EU numbering) that is S; H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0123] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set 404" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is D and the amino acid at position 38 (Kabat numbering) of VL1 is K; ii) CH1 H1 and iii) CH1 H1 and CL1 has an amino acid at position 137 (EU numbering) that is K; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is K and VL2 has an amino acid at position 38 (Kabat numbering) that is D; ii) CH2 H1 and CL2, in which the amino acid at position 147 (EU numbering) is D and the amino acid at position 180 (EU numbering) of CH2 is R; H1and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0124] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set 406" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is E; ii) CH1 H1 and iii) CH1 H1 and CL1 has an amino acid at position 137 (EU numbering) that is K; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is D and VL2 has an amino acid at position 38 (Kabat numbering) that is K; ii) CH2 H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0125] In certain embodiments, the antibody variant comprises "Light Chain Pairing Mutation Set 473" comprising the following substitutions: a) H1 and L1 are: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 and iii) CH1 H1and CL1 has an amino acid at position 137 (EU numbering) that is K; b) H2 and L2 include: i) VH2 has an amino acid at position 39 (Kabat numbering) that is D and VL2 has an amino acid at position 38 (Kabat numbering) that is K; ii) CH2 H1 and CL2, in which the amino acid at position 187 (EU numbering) is D and the amino acid at position 138 (EU numbering) of CH2 is K; H1 and iv) the amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

[0126] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set Q" that includes the following substitutions: a) H1 and L1 are as follows: i) the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 and the amino acid at position 131 (EU numbering) of CL1 is D; H1 and iv) the amino acid at position 220 (EU numbering) in H1H is S and the amino acid at position 214 (EU numbering) in CL1 is S; b) H2 and L2 include: i) VH2 where the amino acid at position 39 (Kabat numbering) is D and the amino acid at position 38 (Kabat numbering) in VL2 is K; and ii) CH2 where the amino acid at position 220 (EU numbering) in H1H is S and the amino acid at position 214 (EU numbering) in CL1 is S. H1 and the amino acid at position 180 (EU numbering) of CL2 is R.

[0127] For example, it may be desirable to modify the antibodies disclosed herein with respect to effector function to enhance the effectiveness of the antibodies in treating diseases and disorders. For example, cysteine ​​residue(s) can be introduced into the Fc region to allow interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922. (1992)). Alternatively, antibodies can be engineered with dual Fc regions, which may therefore have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0128] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0129] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0130] In some embodiments, changes are made in the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0131] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), responsible for maternal-fetal transfer of IgG (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), have been described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0132] In some embodiments, the antibody may comprise a substitution mutation in the Fc region that reduces effector function. In some embodiments, the substitution mutation is a non-glycosylation site mutation. In some embodiments, the non-glycosylation site mutation is at amino acid residue 297 and the amino acid substitutions that disrupt the Fc receptor binding interface are at residues 234, 235, 265, and 331 (EU numbering). In some embodiments, the non-glycosylation site mutation reduces effector function of the antibody.

[0133] In some embodiments, i) CH1 H3 and / or CH2 H3 has A at position 297 (EU numbering), ii) CH1 H3 and / or CH2 H3 has G at position 297 (EU numbering), or iii) CH1 H3 and / or CH2 H3 has an S at position 297 (EU numbering). H3and / or CH2 H3 has S at position 331 (EU numbering).

[0134] In some embodiments, i) H1H and / or H2H have A at positions 234 and 235 (EU numbering), or ii) H1H and / or H2H have A at positions 234, 235 and 237 (EU numbering), or iii) H1H and / or H2H have A at positions 234 and 235 and G at position 329 (EU numbering).

[0135] In some embodiments, the antibody may include substitution mutations in the Fc region that can improve expression titers and increase antibody homogeneity after purification. In some embodiments, the antibody includes a variant human IgG4 Fc domain. In some embodiments, the antibody includes: i) a CH1 H2 and / or CH2 H2 has C at position 370 (Kabat numbering), and ii) CH1 H2 and / or CH2 H2 has a C at position 375 (Kabat numbering).

[0136] The use of knobs-into-holes as a method for producing multispecific antibodies is well known in the art. See U.S. Patent No. 5,731,168 (assigned March 24, 1998 to Genentech), PCT Publication No. WO2009089004 (published July 16, 2009 and assigned to Amgen), and U.S. Patent Publication No. 20090182127 (published July 16, 2009 and assigned to Novo Nordisk A / S). See also Marvin and Zhu, Acta Pharmacologica Sincia (2005) 26(6):649-658 and Kontermann (2005) Acta Pharacol. Sin., 26:1-9.

[0137] A "bulge" refers to at least one amino acid side chain that protrudes from the interface of a first polypeptide and can therefore be positioned in a compensatory cavity in an adjacent interface (i.e., the interface of a second polypeptide), thereby stabilizing, for example, a heteromultimeric antibody, thereby favoring heteromultimeric antibody formation over homomultimeric antibody formation. The bulge may be present in the original interface or may be synthetically introduced (e.g., by modifying the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the first polypeptide is modified to encode a bulge. To achieve this, a nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide is replaced with a nucleic acid encoding at least one "import" amino acid residue having a side chain volume larger than the original amino acid residue. It will be understood that there may be more than one original residue and the corresponding imported residue. The upper limit for the number of original residues that can be replaced is the total number of residues in the interface of the first polypeptide.

[0138] Preferred import residues for forming the bulge are generally naturally occurring amino acid residues, preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Tryptophan and tyrosine are most preferred. In one embodiment, the original residue for forming the bulge has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. The CH1 residue for forming the bulge is preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Tryptophan and tyrosine are most preferred. In one embodiment, the original residue for forming the bulge has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. H3 or CH2 H3 Exemplary amino acid substitutions in the domain include, but are not limited to, a T366W substitution.

[0139] A "cavity" refers to at least one amino acid side chain that is recessed from the interface of a second polypeptide and thus accommodates a corresponding protuberance on the interface of an adjacent first polypeptide. The cavity may be present in the original interface or may be synthetically introduced (e.g., by modifying the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the second polypeptide is modified to encode the cavity. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one "import" amino acid residue having a smaller side chain volume than the original amino acid residue. It will be understood that there may be more than one original residue and the corresponding imported residue. The upper limit for the number of original residues that can be replaced is the total number of residues in the interface of the second polypeptide. The side chain volumes of various amino acid residues are shown in Table 3 above. The imported residues preferred for forming the cavity are typically naturally occurring amino acid residues, preferably selected from alanine (A), serine (S), threonine (T), and valine (V). Most preferred are serine, alanine, or threonine. In one embodiment, the original residue for forming the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. H3 or CH2 H3 Exemplary amino acid substitutions in the domains include, but are not limited to, T366S, L368A, Y407A, Y407T and Y407V substitutions. In certain embodiments, the knob half antibody comprises a T366W substitution and the hole half antibody comprises a T366S / L368A / Y407V substitution.

[0140] In certain embodiments, the antibody variant comprises the following substitution: CH1 H3 has C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering), CH2 H3 but has a C at position 354 and a W at position 366 (EU numbering).

[0141] In certain embodiments, the antibody variant comprises the following substitution: H3has C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering), CH1 H3 but has a C at position 354 and a W at position 366 (EU numbering).

[0142] In certain embodiments, the antibody variant comprises the following substitution: CH1 H3 has C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering), CH2 H3 but has a C at position 349 and a W at position 366 (EU numbering).

[0143] In certain embodiments, the antibody variant comprises the following substitution: H3 has C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering), CH1 H3 but has a C at position 349 and a W at position 366 (EU numbering).

[0144] T cell surface antigen

[0145] The present disclosure provides an antibody comprising a first antigen binding domain that binds to a cell surface antigen expressed on a T cell, a NK cell, a neutrophil, a B cell, or a dendritic cell engager cell, and a second antigen binding domain that binds to a disease associated antigen (DAA). In some embodiments, the cell surface antigen is expressed on a T cell. Exemplary T cell surface antigens include, but are not limited to, CD3. In some embodiments, the T cell surface antigen is CD3. In some embodiments, the T cell surface antigen is CD3ε.

[0146] Differentiation antigen group 3 (CD3)

[0147] In some embodiments, the present invention is based in part on anti-CD3 antibodies. In certain embodiments, the anti-CD3 antibodies are multispecific (e.g., bispecific) and bind to a second biological molecule (e.g., a cell surface antigen, e.g., a disease-associated antigen) in addition to CD3 or a fragment thereof. The antibodies of the present invention are useful, for example, for treating or delaying the progression of cell proliferative disorders (e.g., cancer) or autoimmune disorders, or for enhancing immune function in subjects with such disorders.

[0148] The term "cluster of differentiation 3" or "CD3", as used herein, refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise specified, including, for example, CD3ε, CD3γ, CD3α, and CD3β chains. CD3 is a cell surface complex expressed on T cells in association with the T cell receptor. The CD3 complex is required for the activation of CD8+ and CD4+ T lymphocytes. It is formed from three distinct but highly related chains, one CD3 gamma chain, one CD3 delta chain, and two CD3 epsilon chains, which associate with each other to form the CD3 epsilon / gamma heterodimer and the CD3 epsilon / delta heterodimer. The two CD3 heterodimers, together with the T cell receptor (TCR) and the signaling zeta chain homodimer, form the T cell receptor complex.

[0149] The term encompasses not only "full-length" unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), but also any form of CD3 that results from intracellular processing. The term also encompasses naturally occurring CD3 variants, including, for example, splice variants or allelic variants. CD3 includes, for example, the human CD3ε protein (NCBI RefSeq No. NP_000724), which is 207 amino acids long.

[0150] In some embodiments, the invention provides isolated antibodies that bind to CD3. In some embodiments, the invention provides antibodies that bind to CD3ε. Optionally, the anti-CD3ε antibody binds to a human CD3ε polypeptide or a cynomolgus monkey (cyno) CD3ε polypeptide. Optionally, the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3ε polypeptide (SEQ ID NO: 419) or a cyno CD3ε polypeptide (SEQ ID NO: 420), respectively. Optionally, the anti-CD3 antibody binds to an epitope within a fragment of CD3ε (e.g., human CD3ε) consisting of amino acid residues 1-26 or amino acid residues 1-27 of human CD3ε (SEQ ID NO: 419).

[0151] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group among the target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at the location of the amino acid that shows functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact and neighboring residues may be targeted or eliminated as candidates for substitution. The variants may be screened to determine whether they contain the desired properties.

[0152] Anti-CD3ε antibody

[0153] Provided herein are anti-CD3ε antibodies. In some embodiments, the "SP34" anti-CD3ε antibody was subjected to alanine scanning mutagenesis to generate affinity-tuned anti-CD3ε antibodies of the invention.

[0154] In some embodiments, an anti-CD3ε antibody of the disclosure comprises any one of the VH and VL sequences listed in Table 7. In Table 7, the underlined sequences are the CDR sequences according to Kabat and the bolded sequences are the CDR sequences according to Chothia.

[0155] In some embodiments, the anti-CD3 antibodies of the present disclosure comprise a) a VH complementarity determining region 1 (VH CDR1 ), VH complementarity determining region 2 (VH CDR2 ), and VH complementarity determining region 3 (VH CDR3 ), and b) a heavy chain variable region (VH) comprising a VL complementarity determining region 1 (VL CDR1 ), VL complementarity determining region 2 (VL CDR2 ), and VL complementarity determining region 3 (VL CDR3 ) and a light chain variable region (VL) comprising:

[0156] [Table 9-1] [Table 9-2] [Table 9-3]

[0157] [Table 10-1] [Table 10-2]

[0158] [Table 11]

[0159] In some embodiments, the disclosure provides an antibody (including, for example, an antibody fragment, such as a single chain variable fragment (scFv) that specifically binds to CD3ε), comprising: a) a heavy chain variable region (VH) comprising: i) a VH complementarity determining region 1 (VH complementarity determining region 2) comprising the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, or 33; CDR1 ii) a VH complementarity determining region 2 (VH CDR2 iii) a VH complementarity determining region 3 (VH CDR3 and b) a light chain variable region (VL) comprising i) a VL complementarity determining region 1 (VL) comprising the amino acid sequence of SEQ ID NO: 42. CDR1 ii) a VL complementarity determining region 2 (VL CDR2 iii) a VL complementarity determining region 3 (VL CDR3 ).

[0160] Exemplary anti-CD3 antibodies of the invention include CD3-A1, CD3-A2, CD3-A3, CD3-A4, CD3-A5, CD3-A6, CD3-A7, CD3-A8, CD3-A9, CD3-A10, CD3-A11, CD3-A12, and CD3-A13.

[0161] In some embodiments, the anti-CD3 antibody CD3-A1 comprises a VH comprising the amino acid sequence of SEQ ID NO:29. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:37. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 46 CDR3 The VL region comprises the

[0162] In some embodiments, the anti-CD3 antibody CD3-A1 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:13 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:25.

[0163] In some embodiments, the anti-CD3 antibody CD3-A2 comprises a VH comprising the amino acid sequence of SEQ ID NO:29. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:37. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 44 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 The VL region comprises the

[0164] In some embodiments, the anti-CD3 antibody CD3-A2 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:13 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:27.

[0165] In some embodiments, the anti-CD3 antibody CD3-A3 comprises a VH comprising the amino acid sequence of SEQ ID NO:29. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:37. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 The VL region comprises the

[0166] In some embodiments, the anti-CD3 antibody CD3-A3 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:14 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:23.

[0167] In some embodiments, the anti-CD3 antibody CD3-A4 comprises a VH comprising the amino acid sequence of SEQ ID NO:29. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 34CDR2 and VH comprising the amino acid sequence of SEQ ID NO:38. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 47. CDR3 The VL region comprises the

[0168] In some embodiments, the anti-CD3 antibody CD3-A4 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:15 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:26.

[0169] In some embodiments, the anti-CD3 antibody CD3-A5 comprises a VH comprising the amino acid sequence of SEQ ID NO:29. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:39. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 47. CDR3 The VL region comprises the

[0170] In some embodiments, the anti-CD3 antibody CD3-A5 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:16 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:26.

[0171] In some embodiments, the anti-CD3 antibody CD3-A6 comprises a VH comprising the amino acid sequence of SEQ ID NO: 30. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:37. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 The VL region comprises the

[0172] In some embodiments, the anti-CD3 antibody CD3-A6 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:17 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:22.

[0173] In some embodiments, the anti-CD3 antibody CD3-A7 comprises a VH comprising the amino acid sequence of SEQ ID NO:29. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 35 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:38. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 The VL region comprises the

[0174] In some embodiments, the anti-CD3 antibody CD3-A7 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:18 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:22.

[0175] In some embodiments, the anti-CD3 antibody CD3-A8 comprises a VH comprising the amino acid sequence of SEQ ID NO:29. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 35 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:38. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 47. CDR3 The VL region comprises the

[0176] In some embodiments, the anti-CD3 antibody CD3-A8 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:18 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:26.

[0177] In some embodiments, the anti-CD3 antibody CD3-A9 comprises a VH comprising the amino acid sequence of SEQ ID NO:29. CDR1, a VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and VH comprising the amino acid sequence of SEQ ID NO: 40. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 47. CDR3 The VL region comprises the

[0178] In some embodiments, the anti-CD3 antibody CD3-A9 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:19 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:26.

[0179] In some embodiments, the anti-CD3 antibody CD3-A10 comprises a VH comprising the amino acid sequence of SEQ ID NO:29. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and VH comprising the amino acid sequence of SEQ ID NO: 41. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 47. CDR3 The VL region comprises the

[0180] In some embodiments, the anti-CD3 antibody CD3-A10 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:20 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:26.

[0181] In some embodiments, the anti-CD3 antibody CD3-A11 comprises a VH comprising the amino acid sequence of SEQ ID NO:31. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:37. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 The VL region comprises the

[0182] In some embodiments, the anti-CD3 antibody CD3-A11 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:21 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:22.

[0183] In some embodiments, the anti-CD3 antibody CD3-A12 comprises a VH comprising the amino acid sequence of SEQ ID NO:29. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:37. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 The VL region comprises the

[0184] In some embodiments, the anti-CD3 antibody CD3-A12 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:13 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:24.

[0185] In some embodiments, the anti-CD3 antibody CD3-A13 comprises a VH comprising the amino acid sequence of SEQ ID NO:29. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:39. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 42. CDR1 , VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 48. CDR3 The VL region comprises the

[0186] In some embodiments, the anti-CD3 antibody CD3-A13 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:16 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:28.

[0187] Bispecific anti-CD3ε antibody

[0188] Provided herein are bispecific antibodies comprising a first antigen-binding domain that binds to a first antigen (e.g., CD3ε) and a second antigen-binding domain that binds to a second antigen (e.g., a disease-associated antigen).

[0189] In some embodiments, a bispecific antibody has the following structure: a variable region (VH1), and a constant region 1 domain (CH1 H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domains (CH1 H3 A first heavy chain polypeptide (H1) including a constant region (CH1) having a variable region (VL1) and a constant region (CL1), a first light chain polypeptide (L1) including a variable region (VH2), and a constant region 1 domain (CH2 H1 ), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3 and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).

[0190] In some embodiments, a bispecific antibody of the disclosure comprises a first antigen-binding domain (e.g., that binds CD3) that comprises any one of the VH1 and VL1 sequences listed in Table 7. In Table 7, the underlined sequences are the CDR sequences according to Kabat and the bolded sequences are the CDR sequences according to Chothia.

[0191] In some embodiments, the bispecific antibody of the present disclosure comprises a) a VH complementarity determining region 1 (VH1 CDR1 ), VH complementarity determining region 2 (VH1 CDR2 ), and VH complementarity determining region 3 (VH1 CDR3 ), and b) a heavy chain variable region (VH1) comprising a VL complementarity determining region 1 (VL1 CDR1 ), VL complementarity determining region 2 (VL1 CDR2 ), and VL complementarity determining region 3 (VL1 CDR3) (SEQ ID NO: 1). Tables 8 and 9 provide examples of CDR sequences for anti-CD3 antibodies provided herein.

[0192] In some embodiments, the bispecific antibody comprises any one of the anti-CD3 antibodies of the present disclosure. Exemplary anti-CD3 antibodies of the present invention include CD3-A1, CD3-A2, CD3-A3, CD3-A4, CD3-A5, CD3-A6, CD3-A7, CD3-A8, CD3-A9, CD3-A10, CD3-A11, CD3-A12, and CD3-A13.

[0193] In some embodiments, the disclosure provides an isolated antibody (eg, a monospecific or bispecific antibody) that specifically binds to CD3ε and competes with any of the aforementioned antibodies.

[0194] In some embodiments, the invention provides antibodies (e.g., monospecific or bispecific antibodies) that bind to CD3ε and compete with the antibodies described herein, including CD3-A1, CD3-A2, CD3-A3, CD3-A4, CD3-A5, CD3-A6, CD3-A7, CD3-A8, CD3-A9, CD3-A10, CD3-A11, CD3-A12, and CD3-A13.

[0195] In some embodiments, the present invention also provides CDR portions of antibodies to CD3ε antibodies based on CDR contact regions. CDR contact regions are regions of an antibody that confer specificity to the antibody for an antigen. Generally, CDR contact regions include residue positions within the CDRs and Vernier zones that are constrained to maintain the proper loop structure so that the antibody binds to a particular antigen. See, e.g., Makabe et al., J.Biol.Chem.,283:1156-1166,2007. Determining CDR contact regions is well within the skill of one of ordinary skill in the art.

[0196] The binding affinity (K of an anti-CD3ε antibody (e.g., a monospecific or bispecific antibody) of the invention to human CD3ε (e.g., human CD3ε (e.g., (SEQ ID NO: 419)) D ) can be about 0.001 to about 5000 nM.

[0197] In some embodiments, the binding affinity is about 5000 nM, about 4500 nM, about 4000 nM, about 3500 nM, about 3000 nM, about 2500 nM, about 2000 nM, about 1789 nM, about 1583 nM, about 1540 nM, about 1500 nM, about 1490 nM, about 1064 nM, about 1000 nM, about 933 nM, about 89 4nM, approximately 750nM, approximately 705nM, approximately 678nM, approximately 532nM, approximately 500nM, approximately 494nM, approximately 400nM, approximately 349nM, approximately 340nM, approximately 353 nM, approximately 300nM, approximately 250nM, approximately 244nM, approximately 231nM, approximately 225nM, approximately 207nM, approximately 200nM, approximately 186nM, approximately 172nM, approximately 136n M, about 113nM, about 104nM, about 101nM, about 100nM, about 90nM, about 83nM, about 79nM, about 74nM, about 54nM, about 50nM, about 45n M, about 42nM, about 40nM, about 35nM, about 32nM, about 30nM, about 25nM, about 24nM, about 22nM, about 20nM, about 19nM, about 18nM, about 17 nM, about 16 nM, about 15 nM, about 12 nM, about 10 nM, about 9 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.5 nM, about 0.3 nM, about 0.1 nM, about 0.01 nM, or about 0.001 nM.

[0198] In some embodiments, the binding affinity is less than any of about 5000 nM, about 4000 nM, about 3000 nM, about 2000 nM, about 1000 nM, about 900 nM, about 800 nM, about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 30 nM, about 20 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, or about 0.5 nM.

[0199] The binding affinity (K D ) can be about 0.001 to about 5000 nM.

[0200] In some embodiments, the binding affinity is about 5000 nM, about 4500 nM, about 4000 nM, about 3500 nM, about 3000 nM, about 2500 nM, about 2000 nM, about 1789 nM, about 1583 nM, about 1540 nM, about 1500 nM, about 1490 nM, about 1064 nM, about 1000 nM, about 933 nM, about 89 4nM, approximately 750nM, approximately 705nM, approximately 678nM, approximately 532nM, approximately 500nM, approximately 494nM, approximately 400nM, approximately 349nM, approximately 340nM, approximately 353 nM, approximately 300nM, approximately 250nM, approximately 244nM, approximately 231nM, approximately 225nM, approximately 207nM, approximately 200nM, approximately 186nM, approximately 172nM, approximately 136n M, about 113nM, about 104nM, about 101nM, about 100nM, about 90nM, about 83nM, about 79nM, about 74nM, about 54nM, about 50nM, about 45n M, about 42nM, about 40nM, about 35nM, about 32nM, about 30nM, about 25nM, about 24nM, about 22nM, about 20nM, about 19nM, about 18nM, about 17 nM, about 16 nM, about 15 nM, about 12 nM, about 10 nM, about 9 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.5 nM, about 0.3 nM, about 0.1 nM, about 0.01 nM, or about 0.001 nM.

[0201] In some embodiments, the binding affinity is less than any of about 5000 nM, about 4000 nM, about 3000 nM, about 2000 nM, about 1000 nM, about 900 nM, about 800 nM, about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 30 nM, about 20 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, or about 0.5 nM.

[0202] In some embodiments, the disclosure provides a nucleic acid encoding any of the aforementioned isolated anti-CD3ε antibodies (e.g., monospecific or bispecific antibodies). In some embodiments, the disclosure provides a vector comprising such a nucleic acid. In some embodiments, the disclosure provides a host cell comprising such a nucleic acid.

[0203] Disease-associated antigens

[0204] Provided herein are bispecific antibodies having a first antigen-binding domain that binds to a first antigen (e.g., CD3ε) and a second antigen-binding domain that binds to a second antigen (e.g., a cell surface antigen, or DAA).

[0205] In some embodiments, the second biological molecule is a cell surface antigen. In some embodiments, the second biological molecule is a disease-associated antigen. Disease-associated antigens include ACVR1, ADAM21, AGL10, ALPPL2, APCDD1, ASPRV1, BCMA, BMPR1B, CD151, CD19, CD22, CD274, CD276, CD33, CD38, CD47, CD6, CD70, CD74, CD84, CD180, CDCP1, CDH17, CDH3, CDHR2, CDHR5, CEACAM5, CEACAM6, CEACAM7, CELSR1, CLCA2, CLDN1, CLDN18, CLDN6, CNGB1, CNGB3, COL11A1, COL17A1, CRB1, CPSG4, CTAG2, CTAGE4, ​​CXADR, CXCR4, DCBLD2, DCST1, DLL3, DLL4, DPCR1, DSG3, DSG4, DUOX2, EBI3, EFNA4, EGFR, ENTPD1, ​​ENTPD 2, EPCAM, EPHA10, EPHA6, EPHA8, EPHB3, EPS8L1, ERBB2, ERMP1, F11R, FAP, FAT1, FCER2, FCRL3, FER1L6, FGFR2, FLT3, FLVCR1, FN1, FXYD3, GABR A3, GGT2, GGT3P, GJB3, GLG1, GPC1, GPC2, GPNMB, GPC5A, GRIND2D, GUCY2C, HAVCR2, HEPHL1, HHLA1, IGSF3, IGSF9, IL2RB, IL3RA, ITGA2, ITGA6, ITGAV, ITGB4, ITGB6, LCN15, LILRB4, LNPEP, LRFN4, LRRC15, LY6D, LY75, MAL2, MET, MFI2, MICA, MICB, MMP13, MMP14, MPZL2, MS4A1, MSLN, MST1 R, MTDH, MUC1, MUC13, MUC16, MUC17, NAALADL2, NCSTN, NIPAL4, NLGN1, NOTCH3, NOX1, OC90, OR10Q1, OR5l1, PAEP, PANX3, PCDH15, PDCHA9, PCDH B12, PCDHB2, PKD1L1, PODXL, POLR2J2, PROM1, PSMA, PTK7, PVR, PVRL1, PVRL4, RAET1E, RAET1G, RAET1L, ROR1, ROR2, SDC1, SDC4, SDK2, SHISA8,SIGLEC7, SIT1, SLAMF1, SLAMF6, SLAMF7, SLC11A2, SLC12A2, SLC15A1, SLC1A5, SLC22A25, SLC2A9, SLC34A2, SLC38A2, SL C39A4, SLC6A14, SLC7A11, SLC7A3, SLC7A5, SYT8, TAS2R5, TMEM132A, TMPRSS3, TMPRSS4, TMX1, TNFRSF17, TNFRSF21, TNFR SF9, TNFRSF11, TNFRSF15, TNFRSF4, TNFRSF9, TNMD, TP53111, TPBG, TRPC5, TRPV2, TSPAN10, TSPAN8, UGT2A1, UGT3A2, ULBP1, ULBP2, ULBP3, UMODL1, UPK1B, VANGL1, VANGL2, VASN, VMP1, VSIG4, VTCN1, WNT16, YIF1B, and ZNRF4.

[0206] Exemplary disease-associated antigens include, but are not limited to, those shown in Table 22.

[0207] [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4]

[0208] A multispecific antibody is an antibody (e.g., a monoclonal antibody) that has binding specificities for at least two different sites. In some embodiments, the anti-CD3 CD3ε antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). In certain embodiments, the bispecific antibodies can bind to two different epitopes of CD3 (e.g., CD3ε or CD3γ). In certain embodiments, one of the binding specificities is for CD3 (e.g., CD3ε or CD3γ) and the other is for any other antigen (e.g., a second biological molecule, e.g., a cell surface antigen, e.g., a disease-associated antigen). Thus, a bispecific anti-CD3 antibody can have binding specificities for CD3 and a second biological molecule, such as a second biological molecule (e.g., a disease-associated antigen) listed in Table 22 and described in U.S. Publication No. 2010 / 0111856 and PCT Publication No. WO2016204966A1 (each of which is incorporated herein by reference in its entirety).

[0209] In some cases, a cell surface antigen (e.g., a disease-associated antigen) may be expressed in low copy number on a target cell (e.g., a tumor cell). For example, in some cases, a cell surface antigen is expressed or present at less than 35,000 copies per target cell. In some embodiments, a low copy number cell surface antigen is present at 100-35,000 copies per target cell, 100-30,000 copies per target cell, 100-25,000 copies per target cell, 100-20,000 copies per target cell, 100-15,000 copies per target cell, 100-10,000 copies per target cell, 100-5,000 copies per target cell, 100-2,000 copies per target cell, 100-1,000 copies per target cell, or 100-500 copies per target cell. The copy number of a cell surface antigen may be determined, for example, using a standard scratch card plot.

[0210] UL16 binding protein

[0211] Exemplary UL16 binding proteins include, but are not limited to, ULBP1, ULBP2, ULBP3, RAET1E (ULBP4), RAET1G (ULBP5), and RAET1L (ULBP6). Defects in the regulation of ULBP1-6 are associated with a wide range of diseases, from autoimmunity to cancer.

[0212] UL16-binding protein 2 (ULBP2) is a major histocompatibility complex (MHC) class I-associated molecule that binds to the NKG2D receptor on natural killer (NK) cells, triggering the release of multiple cytokines and chemokines that ultimately contribute to NK cell recruitment and activation. The encoded protein undergoes further processing to generate mature proteins that are either membrane-anchored via glycosyl-phosphatidylinositol moieties or secreted. Many malignant cells secrete the encoded protein to escape immune surveillance by NK cells. ULBP2 is widely and differentially expressed in multiple solid tumor indications. In particular, ULBP2 expression in melanoma and breast cancer is associated with poor prognosis and advanced disease.

[0213] Senescence is a stress-induced cellular state that limits tumorigenesis by preventing cell proliferation and promoting immune-mediated clearance of damaged cells through induction of the senescence-associated secretory phenotype (SASP) (Rodier, F. et al. Persistent DNA damage signaling triggers senescence-associated inflammatory cytokine secretion. Nat. Cell Biol. 11, 973-979 (2009)). Senescence has also been implicated in age-related tissue pathology, where accumulation of SASP-positive cells induces tissue inflammation, which can result in tissue dysfunction manifested as arthritis, autoimmunity, diabetes, fibrosis, and delayed wound healing in elderly patients (Childs, BGet al. Senescent cells: an emerging target for diseases of ageing. Nat. Rev. Drug Discov. 16, 718-735 (2017)). SASP-positive cells express a complex assortment of both secreted and cell surface proteins, including immune-activating cytokines, tissue-remodeling matrix metalloproteinases, and cell surface proteins, including MHCI-like NKG2D ligands that mediate recognition and activation of NK and T cell effectors via the NKG2D costimulatory receptor. Together, these proteins promote the clearance of senescent cells. However, other factors, such as age-related decline in immune cell activity and chemotherapy treatment of cancer, accelerate the induction of SASP-positive cells in tissues and limit the clearance of senescent cells by the immune system (Jackola, DR, Ruger, JK & Miller, RA Age-associated changes in human T cell phenotype and function. Aging Clin. Exp. Res. 6, 25-34 (1994); Demaria, M. et al. Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse. Cancer Discov. 7, 165-176 (2017)).Therapeutic strategies to eliminate SASP-positive cells provide an opportunity to complement senescent immune surveillance and alleviate the underlying pathogenesis of many age-related diseases and the lingering side effects of chemotherapy. Furthermore, not only can clearance of senescent cells reduce age-related diseases, but these senolytic drugs also have the potential to extend life span (Baker, DJ et al. Naturally occurring p16Ink4a-positive cells shorten healthy lifespan. Nature 530, 184-189 (2016); Baker, DJ et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479, 232-236 (2011)). ULBP2, an MHCI-like ligand, has emerged as a cell surface protein associated with stress-induced SASP-positive fibroblasts and cancer cells (Sagiv, A. et al. NKG2D ligands mediate immunosurveillance of senescent cells. Aging 8, 328-344 (2016); Ruscetti, M. et al. NK cell-mediated cytotoxicity contributes to tumor control by a cytostatic drug combination. Science 362, 1416-1422 (2018); Munoz, DP et al. Targetable mechanisms driving immunoevasion of persistent senescent cells link chemotherapy-resistant cancer to aging. JCI Insight 5, e124716, 124716 (2019)). In addition to eradicating cancer cells, drugs targeting ULBP2 or ULBP2 / 5 / 6 have the potential to eliminate SASP-positive cells from tissues, improving tissue function to prevent age-related diseases and extend lifespan.

[0214] ULBP2 includes naturally occurring ULBP2 variants, including, for example, splice variants or allelic variants. ULBP2 includes, for example, the human ULBP2 protein (UniProt ID: Q9BZM5), which is 246 amino acids in length.

[0215] In one aspect, the present invention provides an isolated antibody that binds to ULBP2. Optionally, the anti-ULBP2 antibody binds to a human ULBP2 polypeptide or a portion thereof. In some embodiments, the human ULBP2 polypeptide comprises the amino acid sequence of SEQ ID NO:421.

[0216] ULBP5 (RAET1G) includes naturally occurring ULBP5 variants, including, for example, splice variants or allelic variants. ULBP5 includes, for example, the human ULBP5 protein (UniProt ID: Q6H3X3), which is 334 amino acids in length.

[0217] In one aspect, the present invention provides an isolated antibody that binds to ULBP5 (RAET1G). Optionally, the anti-ULBP5 antibody binds to a human ULBP5 polypeptide or a portion thereof. In some embodiments, the human ULBP5 polypeptide comprises the amino acid sequence of SEQ ID NO:422.

[0218] ULBP6 (RAET1L) includes naturally occurring ULBP6 variants, including, for example, splice variants or allelic variants. ULBP6 includes, for example, the human ULBP6 protein (UniProt ID: Q5VY80), which is 246 amino acids in length.

[0219] In one aspect, the present invention provides an isolated antibody that binds to ULBP6 (RAET1L). Optionally, the anti-ULBP6 antibody binds to a human ULBP6 polypeptide or a portion thereof. In some embodiments, the human ULBP6 polypeptide comprises the amino acid sequence of SEQ ID NO:423.

[0220] Anti-ULBP2 / 5 / 6 antibody

[0221] Provided herein are antibodies that bind to anti-ULBP2 / 5 / 6. In some embodiments, the "E12" anti-ULBP2 / 5 / 6 antibody can be subjected to alanine scanning mutagenesis to produce affinity-tuned anti-ULBP2 / 5 / 6 antibodies. Also provided herein are antibodies that bind to anti-ULBP2. In some embodiments, the "A06" anti-ULBP2 antibody can be subjected to alanine scanning mutagenesis to produce affinity-tuned anti-ULBP2 antibodies.

[0222] In some embodiments, an anti-ULBP2 / 5 / 6 antibody of the disclosure comprises any one of the VH and VL sequences listed in Table 10. In Table 10, the underlined sequences are the CDR sequences according to Kabat and the bolded sequences are the CDR sequences according to Chothia.

[0223] In some embodiments, the anti-ULBP2 antibodies of the disclosure comprise a) a VH complementarity determining region 1 (VH CDR1 ), VH complementarity determining region 2 (VH CDR2 ), and VH complementarity determining region 3 (VH CDR3 ), and b) a heavy chain variable region (VH) comprising a VL complementarity determining region 1 (VL CDR1 ), VL complementarity determining region 2 (VL CDR2 ), and VL complementarity determining region 3 (VL CDR3 ) and a light chain variable region (VL) comprising:

[0224] [Table 13]

[0225] [Table 14]

[0226] [Table 15]

[0227] In some embodiments, the disclosure provides an antibody (including, for example, an antibody fragment, such as a single chain variable fragment (scFv) that specifically binds ULBP2), comprising: a) a heavy chain variable region (VH), comprising: i) a VH complementarity determining region 1 (VH) comprising the amino acid sequence of SEQ ID NO: 5 or 6; CDR1 ii) a VH complementarity determining region 2 (VH CDR2 iii) a VH complementarity determining region 3 (VH CDR3 ), and b) a light chain variable region (VL), comprising i) a VL complementarity determining region 1 (VL) comprising the amino acid sequence of SEQ ID NO: 10. CDR1 ii) a VL complementarity determining region 2 (VL CDR2 iii) VL complementarity determining region 3 (VL CDR3 ).

[0228] Exemplary anti-ULBP2 antibodies of the invention include ULBP2-01, ULBP2-02, E12, A06.

[0229] In some embodiments, the anti-ULBP2 antibody ULBP2-01 comprises a VH comprising the amino acid sequence of SEQ ID NO:5. CDR1 , VH comprising the amino acid sequence of SEQ ID NO:7 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:9. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 10. CDR1 , VL comprising the amino acid sequence of SEQ ID NO:11 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 12. CDR3 The VL region comprises:

[0230] In some embodiments, the anti-ULBP2 antibody ULBP2-01 has a VH region comprising the amino acid sequence set forth in SEQ ID NO:2 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:1.

[0231] In some embodiments, the anti-ULBP2 antibody ULBP2-02 comprises a VH comprising the amino acid sequence of SEQ ID NO:5. CDR1 , VH comprising the amino acid sequence of SEQ ID NO:7 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:9. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 10. CDR1 , VL comprising the amino acid sequence of SEQ ID NO:11 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 12. CDR3 The VL region comprises:

[0232] In some embodiments, the anti-ULBP2 antibody ULBP2-02 has a VH region comprising the amino acid sequence set forth in SEQ ID NO:4 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:3.

[0233] In some embodiments, the anti-ULBP2 antibody E12 comprises a VH comprising the amino acid sequence of SEQ ID NO:5. CDR1 , VH comprising the amino acid sequence of SEQ ID NO:7 CDR2 and VH comprising the amino acid sequence of SEQ ID NO:9. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 10. CDR1 , VL comprising the amino acid sequence of SEQ ID NO:11 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 12. CDR3 The VL region comprises:

[0234] In some embodiments, the anti-ULBP2 antibody E12 has a VH region comprising the amino acid sequence set forth in SEQ ID NO:425 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:424.

[0235] In some embodiments, the anti-ULBP2 antibody A06 comprises a VH comprising the amino acid sequence of SEQ ID NO:428. CDR1 , a VH comprising the amino acid sequence of SEQ ID NO: 430 CDR2 and VH comprising the amino acid sequence of SEQ ID NO: 432. CDR3 and a VH region comprising the amino acid sequence of SEQ ID NO: 433. CDR1, VL comprising the amino acid sequence of SEQ ID NO: 434 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 435. CDR3 The VL region comprises:

[0236] In some embodiments, the anti-ULBP2 antibody A06 has a VH region comprising the amino acid sequence set forth in SEQ ID NO:427 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:426.

[0237] Bispecific anti-ULBP2 / 5 / 6 antibody

[0238] Provided herein are bispecific antibodies comprising a first antigen-binding domain that binds to a first antigen (e.g., a cell surface antigen, CD3ε) and a second antigen-binding domain that binds to a second antigen (e.g., ULBP2 / 5 / 6).

[0239] In some embodiments, a bispecific antibody has the following structure: a variable region (VH1), and a constant region 1 domain (CH1 H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domains (CH1 H3 A first heavy chain polypeptide (H1) including a constant region (CH1) having a variable region (VL1) and a constant region (CL1), a first light chain polypeptide (L1) including a variable region (VH2), and a constant region 1 domain (CH2 H1 ), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3 and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).

[0240] In some embodiments, a bispecific antibody of the disclosure comprises a second antigen-binding domain (e.g., binds ULBP2 / 5 / 6) that comprises any one of the VH2 and VL2 sequences listed in Table 10. In Table 10, the underlined sequences are the CDR sequences according to Kabat and the bolded sequences are the CDR sequences according to Chothia.

[0241] In some embodiments, the bispecific antibody of the present disclosure comprises a) a VH complementarity determining region 1 (VH2 CDR1 ), VH complementarity determining region 2 (VH2 CDR2 ), and VH2 complementarity determining region 3 (VH2 CDR3 ), and b) a heavy chain variable region (VH2) comprising a VL complementarity determining region 1 (VL2 CDR1 ), VL complementarity determining region 2 (VL2 CDR2 ), and VL complementarity determining region 3 (VL2 CDR3 and a second antigen-binding domain (e.g., that binds ULBP2) comprising a light chain variable region (VL2) comprising:

[0242] In some embodiments, the bispecific antibody comprises any one of the anti-ULBP2 / 5 / 6 antibodies of the present disclosure. Exemplary anti-ULBP2 / 5 / 6 antibodies of the present disclosure include ULBP2-01, ULBP2-02, and E12. In some embodiments, the bispecific antibody comprises any one of the anti-ULBP2 antibodies of the present disclosure. Exemplary anti-ULBP2 antibodies of the present disclosure include A06.

[0243] In some embodiments, the present disclosure provides an isolated antibody (eg, a monospecific or bispecific antibody) that specifically binds to ULPB2 / 5 / 6 and competes with any of the aforementioned antibodies.

[0244] In some embodiments, the invention provides antibodies (e.g., monospecific or bispecific antibodies) that bind to ULBP2 / 5 / 6 and compete with the antibodies described herein, including ULBP2-01, ULBP2-02, A06, and E12.

[0245] In some embodiments, the present invention also provides CDR portions of antibodies to ULBP2 / 5 / 6 antibodies based on CDR contact regions. CDR contact regions are regions of an antibody that confer specificity to the antibody for an antigen. Generally, CDR contact regions include residue positions within the CDRs and Vernier zones that are constrained to maintain the proper loop structure so that the antibody binds to a particular antigen. See, e.g., Makabe et al., J.Biol.Chem.,283:1156-1166,2007. Determining CDR contact regions is well within the skill of one of ordinary skill in the art.

[0246] The binding affinity (K of a ULBP2 / 5 / 6 antibody (e.g., a monospecific or bispecific antibody) described herein for ULBP2 / 5 / 6 (e.g., human ULBP2 (e.g., (SEQ ID NO: 421), ULBP5 (SEQ ID NO: 422), ULBP6 (SEQ ID NO: 423)). D ) can be about 0.001 to about 5000 nM.

[0247] In some embodiments, the binding affinity is about 5000 nM, about 4500 nM, about 4000 nM, about 3500 nM, about 3000 nM, about 2500 nM, about 2000 nM, about 1789 nM, about 1583 nM, about 1540 nM, about 1500 nM, about 1490 nM, about 1064 nM, about 1000 nM, about 933 nM, about 89 4nM, approximately 750nM, approximately 705nM, approximately 678nM, approximately 532nM, approximately 500nM, approximately 494nM, approximately 400nM, approximately 349nM, approximately 340nM, approximately 353 nM, approximately 300nM, approximately 250nM, approximately 244nM, approximately 231nM, approximately 225nM, approximately 207nM, approximately 200nM, approximately 186nM, approximately 172nM, approximately 136n M, about 113nM, about 104nM, about 101nM, about 100nM, about 90nM, about 83nM, about 79nM, about 74nM, about 54nM, about 50nM, about 45n M, about 42nM, about 40nM, about 35nM, about 32nM, about 30nM, about 25nM, about 24nM, about 22nM, about 20nM, about 19nM, about 18nM, about 17 nM, about 16 nM, about 15 nM, about 12 nM, about 10 nM, about 9 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.5 nM, about 0.3 nM, about 0.1 nM, about 0.01 nM, or about 0.001 nM.

[0248] In some embodiments, the binding affinity is less than any of about 5000 nM, about 4000 nM, about 3000 nM, about 2000 nM, about 1000 nM, about 900 nM, about 800 nM, about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 30 nM, about 20 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, or about 0.5 nM.

[0249] In some embodiments, the disclosure provides a nucleic acid encoding any of the aforementioned isolated anti-ULBP2 / 5 / 6 antibodies (e.g., monospecific or bispecific antibodies). In some embodiments, the disclosure provides a vector comprising such a nucleic acid. In some embodiments, the disclosure provides a host cell comprising such a nucleic acid.

[0250] Exemplary Bispecific Antibodies that Bind CD3ε and ULBP2 / 5 / 6

[0251] Provided herein is a bispecific antibody comprising a first antigen-binding domain that binds to a first antigen (e.g., CD3ε) and a second antigen-binding domain that binds to a second antigen (e.g., ULBP2 / 5 / 6).

[0252] In some embodiments, a bispecific antibody has the following structure: a variable region (VH1), and a constant region 1 domain (CH1 H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domains (CH1 H3 A first heavy chain polypeptide (H1) including a constant region (CH1) having a variable region (VL1) and a constant region (CL1), a first light chain polypeptide (L1) including a variable region (VH2), and a constant region 1 domain (CH2 H1 ), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3 and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).

[0253] In some embodiments, the bispecific antibody of the present disclosure comprises a) a VH complementarity determining region 1 (VH1 CDR1 ), VH complementarity determining region 2 (VH1 CDR2 ) and VH complementarity determining region 3 (VH1 CDR3 ), and b) a heavy chain variable region (VH1) comprising a VL complementarity determining region 1 (VL1 CDR1 ), VL complementarity determining region 2 (VL1CDR2 ) and VL complementarity determining region 3 (VL1 CDR3 a) a first antigen-binding domain (e.g., binds to CD3ε) comprising a light chain variable region (VL) comprising a VH complementarity determining region 1 (VH2 CDR1 ), VH complementarity determining region 2 (VH2 CDR2 ) and VH2 complementarity determining region 3 (VH2 CDR3 ), and b) a heavy chain variable region (VH2) comprising a VL complementarity determining region 1 (VL2 CDR1 ), VL complementarity determining region 2 (VL2 CDR2 ) and VL complementarity determining region 3 (VL2 CDR3 and a second antigen-binding domain (e.g., binds ULBP2 / 5 / 6) comprising a light chain variable region (VL2) comprising a CDR sequence for the anti-CD3ε antibodies provided herein. Tables 8 and 9 provide example CDR sequences for anti-ULBP2 / 5 / 6 antibodies provided herein. Tables 11 and 12 provide example CDR sequences for anti-ULBP2 / 5 / 6 antibodies provided herein.

[0254] In some embodiments, a bispecific antibody of the disclosure comprises a first antigen-binding domain (e.g., binds CD3ε) comprising any one of the VH1 and VL1 sequences listed in Table 7, and a second antigen-binding domain (e.g., binds ULBP2 / 5 / 6) comprising any one of the VH2 and VL2 sequences listed in Table 10.

[0255] In some embodiments, a bispecific antibody of the disclosure comprises a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1), and a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2) comprising any one of the sequences listed in Table 13 and Table 14. The italicized sequences are the heavy and light chain variable regions. The underlined sequences are the CDRs according to Kabat and the bolded sequences are the CDRs according to Chothia.

[0256] In some embodiments, the bispecific antibodies of the disclosure provided herein comprise an H1 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence of a sequence listed in Table 13 and Table 14.

[0257] In some embodiments, the bispecific antibodies of the disclosure provided herein comprise an L1 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence of a sequence listed in Tables 13 and 14.

[0258] In some embodiments, the bispecific antibodies of the disclosure provided herein comprise an H2 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence of a sequence listed in Tables 13 and 14.

[0259] In some embodiments, the bispecific antibodies of the disclosure provided herein comprise an L2 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence of a sequence listed in Tables 13 and 14.

[0260] In some embodiments, the H1 amino acid sequence is numbered according to SEQ ID NO: 439. In some embodiments, the L1 amino acid sequence is numbered according to SEQ ID NO: 438. In some embodiments, the H2 amino acid sequence is numbered according to SEQ ID NO: 437. In some embodiments, the L2 amino acid sequence is numbered according to SEQ ID NO: 436.

[0261]

Table 16-1

Table 16-2

Table 16-3

Table 16-4

Table 16-5

Table 16-6

Table 16-7

Table 16-8

Table 16-9

Table 16-10

Table 16-11

Table 16-12

Table 16-13

Table 16-14

[0262] Exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibodies of the invention include EIP0174, EIP0175, EIP0187, EIP0205, EIP0206, EIP0207, EIP0208, EIP0294, EIP0295, EIP0306, EIP0307, ​​EIP0318, EIP0340, EIP0342, EIP0354, EIP0356, EIP0367, EIP0377, EIP0404, EIP0406, EIP0473, and EIP0598.

[0263] Bispecific antibodies EIP0174, EIP0175, EIP0187, EIP0205, EIP0206, EIP0207, EIP0208, EIP0294, EIP0295, EIP0306, EIP0307, ​​EIP0318, EIP0340, EIP0342, EIP0354, EIP0356, EIP0367, EIP0377, EIP0404, EIP0406, EIP0473, and EIP0598 have a VH1 domain having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 and a VL1 comprising:

[0264] Bispecific antibodies EIP0174, EIP0175, EIP0187, EIP0205, EIP0206, EIP0207, EIP0208, EIP0294, EIP0295, EIP0306, EIP0307, ​​EIP0318, EIP0340, EIP0342, EIP0354, EIP0356, EIP0367, EIP0377, EIP0404, EIP0406, EIP0473, and EIP0598 have the VH2 domain having the amino acid sequence of SEQ ID NO:5. CDR1 , VH2 having the amino acid sequence of SEQ ID NO:7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO:9. CDR3 and a VL2 having the amino acid sequence of SEQ ID NO: 10. CDR1 , VL2 having the amino acid sequence of SEQ ID NO:11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12. CDR3 and a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising a VL2 having the following sequence:

[0265] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0174 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) of CL1 is K, the amino acid at position 137 (EU numbering) of CL1 is D, H1 the amino acid at position 128 (EU numbering) of CL1 is C, the amino acid at position 118 (EU numbering) of CL1 is C, the amino acid at position 220 (EU numbering) of H1H is S, the amino acid at position 214 (EU numbering) of CL1 is S, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0266] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0174 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 the amino acid at position 147 (EU numbering) of CL2 is D, the amino acid at position 180 (EU numbering) of CL2 is R, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0267] In some embodiments, the bispecific antibody EIP0174 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0268] In some embodiments, the bispecific antibody EIP0174 comprises H1 comprising the amino acid sequence of SEQ ID NO:58, L1 comprising the amino acid sequence of SEQ ID NO:57, H2 comprising the amino acid sequence of SEQ ID NO:56, and L2 comprising the amino acid sequence of SEQ ID NO:55.

[0269] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0175 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) in CL1 is K, the amino acid at position 137 (EU numbering) in CL1 is D, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0270] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0175 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 the amino acid at position 147 (EU numbering) of CL2 is D, the amino acid at position 180 (EU numbering) of CL2 is R, H1 the amino acid at position 134 (EU numbering) of CL2 is C, the amino acid at position 116 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0271] In some embodiments, the bispecific antibody EIP0175 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0272] In some embodiments, the bispecific antibody EIP0175 comprises H1 comprising the amino acid sequence of SEQ ID NO: 62, H2 comprising the amino acid sequence of SEQ ID NO: 60, L1 comprising the amino acid sequence of SEQ ID NO: 61, and L2 comprising the amino acid sequence of SEQ ID NO: 59.

[0273] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0187 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) in CL1 is K, the amino acid at position 137 (EU numbering) in CL1 is D, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0274] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0187 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 the amino acid at position 147 (EU numbering) of CL2 is D, the amino acid at position 180 (EU numbering) of CL2 is R, H1 the amino acid at position 136 (EU numbering) of CL2 is C, the amino acid at position 114 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0275] In some embodiments, the bispecific antibody EIP0187 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0276] In some embodiments, the bispecific antibody EIP0187 comprises H1 comprising the amino acid sequence of SEQ ID NO: 66, H2 comprising the amino acid sequence of SEQ ID NO: 64, L1 comprising the amino acid sequence of SEQ ID NO: 65, and L2 comprising the amino acid sequence of SEQ ID NO: 63.

[0277] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0205 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 147 (EU numbering) of CL1 is K, the amino acid at position 131 (EU numbering) of CL1 is D, H1 the amino acid at position 173 (EU numbering) of CL1 is C, the amino acid at position 162 (EU numbering) of CL1 is C, the amino acid at position 220 (EU numbering) of H1H is S, the amino acid at position 214 (EU numbering) of CL1 is S, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 the amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0278] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0205 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 the amino acid at position 147 (EU numbering) of CL2 is D, the amino acid at position 180 (EU numbering) of CL2 is R, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0279] In some embodiments, the bispecific antibody EIP0205 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0280] In some embodiments, the bispecific antibody EIP0205 comprises H1 comprising the amino acid sequence of SEQ ID NO:70, H2 comprising the amino acid sequence of SEQ ID NO:68, L1 comprising the amino acid sequence of SEQ ID NO:69, and L2 comprising the amino acid sequence of SEQ ID NO:67.

[0281] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0206 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) of CL1 is K, the amino acid at position 137 (EU numbering) of CL1 is D, H1the amino acid at position 173 (EU numbering) of CL1 is C, the amino acid at position 162 (EU numbering) of CL1 is C, the amino acid at position 220 (EU numbering) in H1H is S, and the amino acid at position 214 in CL1 is S; The amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A; H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 the amino acid at position 407 (EU numbering) is V; CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0282] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0206 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 the amino acid at position 147 (EU numbering) of CL2 is D, the amino acid at position 180 (EU numbering) of CL2 is R, H1 the amino acid at position 134 (EU numbering) of CL2 is C, the amino acid at position 116 (EU numbering) of CL2 is C, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0283] In some embodiments, the bispecific antibody EIP0206 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0284] In some embodiments, the bispecific antibody EIP0206 comprises H1 comprising the amino acid sequence of SEQ ID NO: 74, H2 comprising the amino acid sequence of SEQ ID NO: 72, L1 comprising the amino acid sequence of SEQ ID NO: 73, and L2 comprising the amino acid sequence of SEQ ID NO: 71.

[0285] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0207 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) in CL1 is K, the amino acid at position 137 (EU numbering) in CL1 is D, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0286] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0207 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1the amino acid at position 147 (EU numbering) of CL2 is D, the amino acid at position 180 (EU numbering) of CL2 is R, H1 the amino acid at position 131 (EU numbering) of CL2 is C, the amino acid at position 114 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0287] In some embodiments, the bispecific antibody EIP0207 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0288] In some embodiments, the bispecific antibody EIP0207 comprises H1 comprising the amino acid sequence of SEQ ID NO: 78, H2 comprising the amino acid sequence of SEQ ID NO: 76, L1 comprising the amino acid sequence of SEQ ID NO: 77, and L2 comprising the amino acid sequence of SEQ ID NO: 75.

[0289] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0208 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) in CL1 is K, the amino acid at position 137 (EU numbering) in CL1 is D, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0290] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0208 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 The amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 138 (EU numbering) of CL2 is K, and H1 the amino acid at position 170 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0291] In some embodiments, the bispecific antibody EIP0208 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0292] In some embodiments, the bispecific antibody EIP0208 comprises H1 comprising the amino acid sequence of SEQ ID NO: 82, H2 comprising the amino acid sequence of SEQ ID NO: 80, L1 comprising the amino acid sequence of SEQ ID NO: 81, and L2 comprising the amino acid sequence of SEQ ID NO: 79.

[0293] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0294 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) in CL1 is E, the amino acid at position 137 (EU numbering) in CL1 is K, the amino acid at position 179 (EU numbering) in CL1 is E, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 the amino acid at position 407 (EU numbering) is V; CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0294] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0294 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 The amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 138 (EU numbering) of CL2 is K, and H1the amino acid at position 171 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0295] In some embodiments, the bispecific antibody EIP0294 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0296] In some embodiments, the bispecific antibody EIP0294 comprises H1 comprising the amino acid sequence of SEQ ID NO: 86, H2 comprising the amino acid sequence of SEQ ID NO: 84, L1 comprising the amino acid sequence of SEQ ID NO: 85, and L2 comprising the amino acid sequence of SEQ ID NO: 83.

[0297] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0295 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) in CL1 is K, the amino acid at position 137 (EU numbering) in CL1 is D, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0298] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0295 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 The amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 138 (EU numbering) of CL2 is K, and H1 the amino acid at position 171 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0299] In some embodiments, the bispecific antibody EIP0295 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0300] In some embodiments, the bispecific antibody EIP0295 comprises H1 comprising the amino acid sequence of SEQ ID NO: 90, H2 comprising the amino acid sequence of SEQ ID NO: 88, L1 comprising the amino acid sequence of SEQ ID NO: 89, and L2 comprising the amino acid sequence of SEQ ID NO: 87.

[0301] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0306 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) of CL1 is E, the amino acid at position 137 (EU numbering) of CL1 is K, the amino acid at position 179 (EU numbering) of CL1 is E, H1 the amino acid at position 185 (EU numbering) in CL1 is E; the amino acid at position 179 (EU numbering) in CL1 is E; the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A; H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 the amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0302] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0306 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 the amino acid at position 147 (EU numbering) of CL2 is D, the amino acid at position 180 (EU numbering) of CL2 is R, H1the amino acid at position 171 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0303] In some embodiments, the bispecific antibody EIP0306 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0304] In some embodiments, the bispecific antibody EIP0306 comprises H1 comprising the amino acid sequence of SEQ ID NO: 94, H2 comprising the amino acid sequence of SEQ ID NO: 92, L1 comprising the amino acid sequence of SEQ ID NO: 93, and L2 comprising the amino acid sequence of SEQ ID NO: 91.

[0305] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0307 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) in CL1 is K, the amino acid at position 137 (EU numbering) in CL1 is D, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0306] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0307 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 the amino acid at position 147 (EU numbering) of CL2 is D, the amino acid at position 180 (EU numbering) of CL2 is R, H1 the amino acid at position 171 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0307] In some embodiments, the bispecific antibody EIP0307 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0308] In some embodiments, the bispecific antibody EIP0307 comprises H1 comprising the amino acid sequence of SEQ ID NO: 98, H2 comprising the amino acid sequence of SEQ ID NO: 96, L1 comprising the amino acid sequence of SEQ ID NO: 97, and L2 comprising the amino acid sequence of SEQ ID NO: 95.

[0309] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0318 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 147 (EU numbering) of CL1 is K, the amino acid at position 131 (EU numbering) of CL1 is D, H1 the amino acid at position 185 (EU numbering) in CL1 is K, the amino acid at position 137 (EU numbering) in CL1 is D, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 the amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0310] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0318 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 the amino acid at position 147 (EU numbering) of CL2 is D, the amino acid at position 180 (EU numbering) of CL2 is R, H1 the amino acid at position 171 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0311] In some embodiments, the bispecific antibody EIP0318 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0312] In some embodiments, the bispecific antibody EIP0318 comprises H1 comprising the amino acid sequence of SEQ ID NO: 102, H2 comprising the amino acid sequence of SEQ ID NO: 100, L1 comprising the amino acid sequence of SEQ ID NO: 101, and L2 comprising the amino acid sequence of SEQ ID NO: 99.

[0313] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0340 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is D, the amino acid at position 38 (Kabat numbering) of VL1 is K, and the amino acid at position 39 (Kabat numbering) of VL1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) in CL1 is E, the amino acid at position 137 (EU numbering) in CL1 is K, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0314] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0340 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is K, the amino acid at position 38 (Kabat numbering) of VL2 is D, and the amino acid at position 39 (Kabat numbering) of VL2 is D, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 The amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 138 (EU numbering) of CL2 is K, and H1 the amino acid at position 136 (EU numbering) of CL2 is C, the amino acid at position 114 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0315] In some embodiments, the bispecific antibody EIP0340 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 14, a VH2 comprising the amino acid sequence of SEQ ID NO: 4, a VL1 comprising the amino acid sequence of SEQ ID NO: 23, and a VL2 comprising the amino acid sequence of SEQ ID NO: 3.

[0316] In some embodiments, the bispecific antibody EIP0340 comprises H1 comprising the amino acid sequence of SEQ ID NO: 106, H2 comprising the amino acid sequence of SEQ ID NO: 104, L1 comprising the amino acid sequence of SEQ ID NO: 105, and L2 comprising the amino acid sequence of SEQ ID NO: 103.

[0317] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0342 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is D, the amino acid at position 38 (Kabat numbering) of VL1 is K, and the amino acid at position 39 (Kabat numbering) of VL1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) in CL1 is E, the amino acid at position 137 (EU numbering) in CL1 is K, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0318] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0342 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is K, the amino acid at position 38 (Kabat numbering) of VL2 is D, and the amino acid at position 39 (Kabat numbering) of VL2 is D, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 The amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 138 (EU numbering) of CL2 is K, and H1 the amino acid at position 171 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0319] In some embodiments, the bispecific antibody EIP0342 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 14, a VH2 comprising the amino acid sequence of SEQ ID NO: 4, a VL1 comprising the amino acid sequence of SEQ ID NO: 23, and a VL2 comprising the amino acid sequence of SEQ ID NO: 3.

[0320] In some embodiments, the bispecific antibody EIP0342 comprises H1 comprising the amino acid sequence of SEQ ID NO:110, H2 comprising the amino acid sequence of SEQ ID NO:108, L1 comprising the amino acid sequence of SEQ ID NO:109, and L2 comprising the amino acid sequence of SEQ ID NO:107.

[0321] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0354 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is D, the amino acid at position 38 (Kabat numbering) of VL1 is K, and the amino acid at position 39 (Kabat numbering) of VL1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 185 (EU numbering) in CL1 is K, the amino acid at position 137 (EU numbering) in CL1 is D, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0322] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0354 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is K, the amino acid at position 38 (Kabat numbering) of VL2 is D, and the amino acid at position 39 (Kabat numbering) of VL2 is D, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 The amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 138 (EU numbering) of CL2 is K, and H1 the amino acid at position 171 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0323] In some embodiments, the bispecific antibody EIP0354 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 14, a VH2 comprising the amino acid sequence of SEQ ID NO: 4, a VL1 comprising the amino acid sequence of SEQ ID NO: 23, and a VL2 comprising the amino acid sequence of SEQ ID NO: 3.

[0324] In some embodiments, the bispecific antibody EIP0354 comprises H1 comprising the amino acid sequence of SEQ ID NO:114, H2 comprising the amino acid sequence of SEQ ID NO:112, L1 comprising the amino acid sequence of SEQ ID NO:113, and L2 comprising the amino acid sequence of SEQ ID NO:111.

[0325] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0356 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is D, the amino acid at position 38 (Kabat numbering) of VL1 is K, and the amino acid at position 39 (Kabat numbering) of VL1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 147 (EU numbering) of CL1 is K, the amino acid at position 131 (EU numbering) of CL1 is D, H1 the amino acid at position 185 (EU numbering) of CL1 is E, the amino acid at position 137 (EU numbering) of CL1 is K, H1 the amino acid at position 145 (EU numbering) in CL1 is S, the amino acid at position 180 (EU numbering) in CL1 is E, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0326] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0356 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is K, the amino acid at position 38 (Kabat numbering) of VL2 is D, and the amino acid at position 39 (Kabat numbering) of VL2 is D, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 The amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 138 (EU numbering) of CL2 is K, and H1the amino acid at position 170 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0327] In some embodiments, the bispecific antibody EIP0356 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 14, a VH2 comprising the amino acid sequence of SEQ ID NO: 4, a VL1 comprising the amino acid sequence of SEQ ID NO: 23, and a VL2 comprising the amino acid sequence of SEQ ID NO: 3.

[0328] In some embodiments, the bispecific antibody EIP0356 comprises H1 comprising the amino acid sequence of SEQ ID NO: 118, H2 comprising the amino acid sequence of SEQ ID NO: 116, L1 comprising the amino acid sequence of SEQ ID NO: 117, and L2 comprising the amino acid sequence of SEQ ID NO: 115.

[0329] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0367 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VL1 is D, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 147 (EU numbering) of CL1 is D, the amino acid at position 131 (EU numbering) of CL1 is K, H1 the amino acid at position 185 (EU numbering) in CL1 is E, the amino acid at position 137 (EU numbering) in CL1 is D, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0330] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0367 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 the amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 137 (EU numbering) of CL2 is K, the amino acid at position 138 (EU numbering) of CL2 is R, H1 the amino acid at position 170 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0331] In some embodiments, the bispecific antibody EIP0367 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0332] In some embodiments, the bispecific antibody EIP0367 comprises H1 comprising the amino acid sequence of SEQ ID NO: 122, H2 comprising the amino acid sequence of SEQ ID NO: 120, L1 comprising the amino acid sequence of SEQ ID NO: 121, and L2 comprising the amino acid sequence of SEQ ID NO: 119.

[0333] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0377 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 The amino acid at position 147 (EU numbering) of CH1 is D, H1 the amino acid at position 145 (EU numbering) in CL1 is S, the amino acid at position 131 (EU numbering) in CL1 is K, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 the amino acid at position 407 (EU numbering) is V; CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0334] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0377 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 The amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 138 (EU numbering) of CL2 is K, and H1the amino acid at position 170 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0335] In some embodiments, the bispecific antibody EIP0377 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0336] In some embodiments, the bispecific antibody EIP0377 comprises H1 comprising the amino acid sequence of SEQ ID NO: 126, H2 comprising the amino acid sequence of SEQ ID NO: 124, L1 comprising the amino acid sequence of SEQ ID NO: 125, and L2 comprising the amino acid sequence of SEQ ID NO: 123.

[0337] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0404 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is D, the amino acid at position 38 (Kabat numbering) of VL1 is K, and the amino acid at position 39 (Kabat numbering) of VL1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 147 (EU numbering) of CL1 is K, the amino acid at position 131 (EU numbering) of CL1 is D, H1 the amino acid at position 185 (EU numbering) in CL1 is E, the amino acid at position 137 (EU numbering) in CL1 is K, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0338] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0404 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is K, the amino acid at position 38 (Kabat numbering) of VL2 is D, and the amino acid at position 39 (Kabat numbering) of VL2 is D, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 the amino acid at position 147 (EU numbering) of CL2 is D, the amino acid at position 180 (EU numbering) of CL2 is R, H1 the amino acid at position 136 (EU numbering) of CL2 is C, the amino acid at position 114 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0339] In some embodiments, the bispecific antibody EIP0404 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 24, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0340] In some embodiments, the bispecific antibody EIP0404 comprises H1 comprising the amino acid sequence of SEQ ID NO: 130, H2 comprising the amino acid sequence of SEQ ID NO: 128, L1 comprising the amino acid sequence of SEQ ID NO: 129, and L2 comprising the amino acid sequence of SEQ ID NO: 127.

[0341] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0406 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is E, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 147 (EU numbering) of CL1 is K, the amino acid at position 131 (EU numbering) of CL1 is D, H1 the amino acid at position 185 (EU numbering) in CL1 is E, the amino acid at position 137 (EU numbering) in CL1 is K, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0342] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0406 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 The amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 138 (EU numbering) of CL2 is K, and H1the amino acid at position 136 (EU numbering) of CL2 is C, the amino acid at position 114 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0343] In some embodiments, the bispecific antibody EIP0406 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 24, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0344] In some embodiments, the bispecific antibody EIP0406 comprises H1 comprising the amino acid sequence of SEQ ID NO: 134, H2 comprising the amino acid sequence of SEQ ID NO: 132, L1 comprising the amino acid sequence of SEQ ID NO: 133, and L2 comprising the amino acid sequence of SEQ ID NO: 131.

[0345] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0473 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 147 (EU numbering) of CL1 is D, the amino acid at position 131 (EU numbering) of CL1 is K, H1 the amino acid at position 185 (EU numbering) in CL1 is D, the amino acid at position 137 (EU numbering) in CL1 is K, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0346] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0473 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1 The amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 138 (EU numbering) of CL2 is K, and H1 the amino acid at position 171 (EU numbering) of CL2 is C, the amino acid at position 162 (EU numbering) of CL2 is C, the amino acid at position 220 (EU numbering) in H2H is S, the amino acid at position 214 (EU numbering) of CL2 is S, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0347] In some embodiments, the bispecific antibody EIP0473 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 4, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 3.

[0348] In some embodiments, the bispecific antibody EIP0473 comprises H1 comprising the amino acid sequence of SEQ ID NO: 138, H2 comprising the amino acid sequence of SEQ ID NO: 136, L1 comprising the amino acid sequence of SEQ ID NO: 137, and L2 comprising the amino acid sequence of SEQ ID NO: 135.

[0349] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0598 contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438. H1 the amino acid at position 170 (EU numbering) of CL1 is S, the amino acid at position 131 (EU numbering) of CL2 is D, H1 the amino acid at position 173 (EU numbering) of CL1 is C, the amino acid at position 162 (EU numbering) of CL1 is C, the amino acid at position 220 (EU numbering) of H1H is S, the amino acid at position 214 (EU numbering) of CL1 is S, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0350] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0598 contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat numbering) of VH2 is D, the amino acid at position 38 (Kabat numbering) of VL2 is K, and the amino acid at position 39 (Kabat numbering) of VL2 is K, when numbered according to the H2 amino acid sequence of SEQ ID NO: 437 and the L2 amino acid sequence of SEQ ID NO: 436. H1the amino acid at position 147 (EU numbering) of CL2 is D, the amino acid at position 180 (EU numbering) of CL2 is R, the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A, and CH2 H3 The amino acid at position 354 (EU numbering) is C, and CH2 H3 The amino acid at position 366 (EU numbering) is W, and CH2 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0351] In some embodiments, the bispecific antibody EIP0598 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0352] In some embodiments, the bispecific antibody EIP0598 comprises H1 comprising the amino acid sequence of SEQ ID NO: 142, H2 comprising the amino acid sequence of SEQ ID NO: 140, L1 comprising the amino acid sequence of SEQ ID NO: 141, and L2 comprising the amino acid sequence of SEQ ID NO: 139.

[0353] Any one of the bispecific antibodies shown in Table 13 above can be further modified by replacing any one of the anti-CD3ε antigen binding regions with any one of the anti-CD3ε binding regions shown in Tables 7-9. For example, the anti-CD3ε antigen binding region of the bispecific antibody "EIP0205" can be replaced with any one of the anti-CD3ε binding regions shown in Tables 7-9 to produce a bispecific antibody of the invention. Exemplary antibodies are shown in Table 14. The underlined sequences are the CDR sequences according to Kabat and the bolded sequences are the CDR sequences according to Chothia.

[0354] [Table 17-1] [Table 17-2]

Table 17-3

Table 17-4

Table 17-5

Table 17-6

Table 17-7

Table 17-8

Table 17-9

Table 17-10

Table 17-11

Table 17-12

Table 17-13

Table 17-14

Table 17-15

Table 17-16

[0355] In some embodiments, exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibodies of the invention include EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, EIP0525.

[0356] Bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525 contain the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438; CH1 H1 the amino acid at position 147 (EU numbering) of CL1 is K, the amino acid at position 131 (EU numbering) of CL1 is D, H1 the amino acid at position 173 (EU numbering) of CL1 is C, the amino acid at position 162 (EU numbering) of CL1 is C, the amino acid at position 220 (EU numbering) of H1H is S, the amino acid at position 214 (EU numbering) of CL1 is S, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0357] The bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525 have the VH2 domain having the amino acid sequence of SEQ ID NO:5. CDR1 , VH2 having the amino acid sequence of SEQ ID NO:7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO:9. CDR3 and a VL2 having the amino acid sequence of SEQ ID NO: 10. CDR1 , VL2 having the amino acid sequence of SEQ ID NO:11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12. CDR3 and a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising a VL2 comprising:

[0358] Bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525 comprise a VH2 comprising the amino acid sequence of SEQ ID NO:2 and a VL2 comprising the amino acid sequence of SEQ ID NO:1.

[0359] Bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525 have H2 comprising the amino acid sequence of SEQ ID NO:68 and L2 comprising the amino acid sequence of SEQ ID NO:67.

[0360] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0527 comprises a VH1 having the amino acid sequence of SEQ ID NO:29.CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 46 CDR3 and a VL1 comprising:

[0361] In some embodiments, the bispecific antibody EIP0527 comprises a VH1 having the amino acid sequence of SEQ ID NO: 13 and a VL1 having the amino acid sequence of SEQ ID NO: 25. In some embodiments, the bispecific antibody EIP0527 comprises an H1 having the amino acid sequence of SEQ ID NO: 146 and an L1 having the amino acid sequence of SEQ ID NO: 145.

[0362] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0624 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO:38 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 and a VL1 comprising:

[0363] In some embodiments, the bispecific antibody EIP0624 comprises a VH1 having the amino acid sequence of SEQ ID NO: 15 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0624 comprises an H1 having the amino acid sequence of SEQ ID NO: 150 and an L1 having the amino acid sequence of SEQ ID NO: 149.

[0364] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0486 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 44 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 and a VL1 comprising:

[0365] In some embodiments, the bispecific antibody EIP0486 comprises a VH1 having the amino acid sequence of SEQ ID NO: 13 and a VL1 having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the bispecific antibody EIP0486 comprises an H1 having the amino acid sequence of SEQ ID NO: 154 and an L1 having the amino acid sequence of SEQ ID NO: 153.

[0366] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0626 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO:39 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 and a VL1 comprising:

[0367] In some embodiments, the bispecific antibody EIP0626 comprises a VH1 having the amino acid sequence of SEQ ID NO: 16 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0626 comprises an H1 having the amino acid sequence of SEQ ID NO: 158 and an L1 having the amino acid sequence of SEQ ID NO: 157.

[0368] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0483 comprises a VH1 having the amino acid sequence of SEQ ID NO: 30. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 and a VL1 comprising:

[0369] In some embodiments, the bispecific antibody EIP0483 comprises a VH1 having the amino acid sequence of SEQ ID NO: 17 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0483 comprises an H1 having the amino acid sequence of SEQ ID NO: 162 and an L1 having the amino acid sequence of SEQ ID NO: 161.

[0370] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0623 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO:38 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3and a VL1 comprising:

[0371] In some embodiments, the bispecific antibody EIP0623 comprises a VH1 having the amino acid sequence of SEQ ID NO: 18 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0623 comprises an H1 having the amino acid sequence of SEQ ID NO: 166 and an L1 having the amino acid sequence of SEQ ID NO: 165.

[0372] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0621 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 40. CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 and a VL1 comprising:

[0373] In some embodiments, the bispecific antibody EIP0621 comprises a VH1 having the amino acid sequence of SEQ ID NO: 19 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0621 comprises an H1 having the amino acid sequence of SEQ ID NO: 170 and an L1 having the amino acid sequence of SEQ ID NO: 169.

[0374] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0625 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO:38 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1, VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 and a VL1 comprising:

[0375] In some embodiments, the bispecific antibody EIP0625 comprises a VH1 having the amino acid sequence of SEQ ID NO: 18 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0625 comprises an H1 having the amino acid sequence of SEQ ID NO: 174 and an L1 having the amino acid sequence of SEQ ID NO: 173.

[0376] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0622 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 41. CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 and a VL1 comprising:

[0377] In some embodiments, the bispecific antibody EIP0622 comprises a VH1 having the amino acid sequence of SEQ ID NO: 20 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0622 comprises an H1 having the amino acid sequence of SEQ ID NO: 178 and an L1 having the amino acid sequence of SEQ ID NO: 177.

[0378] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0525 comprises a VH1 having the amino acid sequence of SEQ ID NO:31. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 and a VL1 comprising:

[0379] In some embodiments, the bispecific antibody EIP0525 comprises a VH1 having the amino acid sequence of SEQ ID NO: 21 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0525 comprises an H1 having the amino acid sequence of SEQ ID NO: 182 and an L1 having the amino acid sequence of SEQ ID NO: 181.

[0380] In some embodiments, exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibodies of the invention include EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, EIP0629.

[0381] Bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629 have the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, the amino acid at position 38 (Kabat numbering) of VL1 is D, and the amino acid at position 39 (Kabat numbering) of VH1 is K, when numbered according to the H1 amino acid sequence of SEQ ID NO:439 and the L1 amino acid sequence of SEQ ID NO:438. H1 the amino acid at position 170 (EU numbering) of CL1 is S, the amino acid at position 131 (EU numbering) of CL2 is D, H1the amino acid at position 173 (EU numbering) of CL1 is C, the amino acid at position 162 (EU numbering) of CL1 is C, the amino acid at position 220 (EU numbering) of H1H is S, the amino acid at position 214 (EU numbering) of CL1 is S, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0382] The bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629 have the VH2 amino acid sequence of SEQ ID NO:5. CDR1 , VH2 comprising the amino acid sequence of SEQ ID NO:7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO:9. CDR3 and VL2 having the amino acid sequence of SEQ ID NO: 10. CDR1 , VL2 having the amino acid sequence of SEQ ID NO:11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12. CDR3 and a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising a VL2 having the following structure:

[0383] Bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629 have a VH2 having the amino acid sequence of SEQ ID NO:2 and a VL2 having the amino acid sequence of SEQ ID NO:1.

[0384] Bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629 have an H2 having the amino acid sequence of SEQ ID NO: 140 and an L2 having the amino acid sequence of SEQ ID NO: 139.

[0385] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0630 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 46 CDR3 and a VL1 comprising:

[0386] In some embodiments, the bispecific antibody EIP0630 comprises a VH1 having the amino acid sequence of SEQ ID NO: 13 and a VL1 having the amino acid sequence of SEQ ID NO: 25. In some embodiments, the bispecific antibody EIP0630 comprises an H1 having the amino acid sequence of SEQ ID NO: 186 and an L1 having the amino acid sequence of SEQ ID NO: 185.

[0387] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0540 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO:38 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3and a VL1 comprising:

[0388] In some embodiments, the bispecific antibody EIP0540 comprises a VH1 having the amino acid sequence of SEQ ID NO: 15 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0540 comprises an H1 having the amino acid sequence of SEQ ID NO: 190 and an L1 having the amino acid sequence of SEQ ID NO: 189.

[0389] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0628 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 44 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 and a VL1 comprising:

[0390] In some embodiments, the bispecific antibody EIP0628 comprises a VH1 having the amino acid sequence of SEQ ID NO: 13 and a VL1 having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the bispecific antibody EIP0628 comprises an H1 having the amino acid sequence of SEQ ID NO: 194 and an L1 having the amino acid sequence of SEQ ID NO: 193.

[0391] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0542 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO:39 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1, VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 and a VL1 comprising:

[0392] In some embodiments, the bispecific antibody EIP0542 comprises a VH1 having the amino acid sequence of SEQ ID NO: 16 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0542 comprises an H1 having the amino acid sequence of SEQ ID NO: 198 and an L1 having the amino acid sequence of SEQ ID NO: 197.

[0393] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0627 comprises a VH1 having the amino acid sequence of SEQ ID NO: 30. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 and a VL1 comprising:

[0394] In some embodiments, the bispecific antibody EIP0627 comprises a VH1 having the amino acid sequence of SEQ ID NO: 17 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0627 comprises an H1 having the amino acid sequence of SEQ ID NO: 202 and an L1 having the amino acid sequence of SEQ ID NO: 201.

[0395] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0515 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO:38CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 and a VL1 comprising:

[0396] In some embodiments, the bispecific antibody EIP0515 comprises a VH1 having the amino acid sequence of SEQ ID NO: 18 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0515 comprises an H1 having the amino acid sequence of SEQ ID NO: 206 and an L1 having the amino acid sequence of SEQ ID NO: 205.

[0397] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0477 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 40. CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 and a VL1 comprising:

[0398] In some embodiments, the bispecific antibody EIP0477 comprises a VH1 having the amino acid sequence of SEQ ID NO: 19 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0477 comprises an H1 having the amino acid sequence of SEQ ID NO: 210 and an L1 having the amino acid sequence of SEQ ID NO: 209.

[0399] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0541 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1, VH1 having the amino acid sequence of SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO:38 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 and a VL1 comprising:

[0400] In some embodiments, the bispecific antibody EIP0541 comprises a VH1 having the amino acid sequence of SEQ ID NO: 18 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0541 comprises an H1 having the amino acid sequence of SEQ ID NO: 214 and an L1 having the amino acid sequence of SEQ ID NO: 213.

[0401] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0513 comprises a VH1 having the amino acid sequence of SEQ ID NO:29. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 41. CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 and a VL1 comprising:

[0402] In some embodiments, the bispecific antibody EIP0513 comprises a VH1 having the amino acid sequence of SEQ ID NO: 20 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0513 comprises an H1 having the amino acid sequence of SEQ ID NO: 218 and an L1 having the amino acid sequence of SEQ ID NO: 217.

[0403] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0629 comprises a VH1 having the amino acid sequence of SEQ ID NO:31. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 and a VL1 comprising:

[0404] In some embodiments, the bispecific antibody EIP0629 comprises a VH1 having the amino acid sequence of SEQ ID NO: 21 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0629 comprises an H1 having the amino acid sequence of SEQ ID NO: 222 and an L1 having the amino acid sequence of SEQ ID NO: 221.

[0405] In some embodiments, exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibodies of the invention include EIP0820.

[0406] In some embodiments, the bispecific antibody EIP0820 comprises the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K and the amino acid at position 38 (Kabat numbering) of VL1 is D, when numbered according to the H1 amino acid sequence of SEQ ID NO: 439 and the L1 amino acid sequence of SEQ ID NO: 438; CH1 H1 the amino acid at position 147 (EU numbering) of CL1 is K, the amino acid at position 131 (EU numbering) of CL1 is D, H1the amino acid at position 173 (EU numbering) of CL1 is C, the amino acid at position 162 (EU numbering) of CL1 is C, the amino acid at position 220 (EU numbering) of H1H is S, the amino acid at position 214 (EU numbering) of CL1 is S, the amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A, H3 The amino acid at position 349 (EU numbering) is C, and CH1 H3 The amino acid at position 366 (EU numbering) is S, and CH1 H3 The amino acid at position 368 (EU numbering) is A, and CH1 H3 The amino acid at position 407 (EU numbering) is V, and CH1 H3 There is a deletion of the amino acid at position 447 (EU numbering).

[0407] In some embodiments, the bispecific antibody EIP0820 has a VH2 having the amino acid sequence of SEQ ID NO:5. CDR1 , VH2 having the amino acid sequence of SEQ ID NO:7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO:9. CDR3 and a VL2 having the amino acid sequence of SEQ ID NO: 10. CDR1 , VL2 having the amino acid sequence of SEQ ID NO:11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12. CDR3 and a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising a VL2 comprising:

[0408] In some embodiments, the bispecific antibody EIP0820 comprises a VH2 comprising the amino acid sequence of SEQ ID NO:2 and a VL2 comprising the amino acid sequence of SEQ ID NO:1.

[0409] In some embodiments, the bispecific antibody EIP0820 comprises a VH2 comprising the amino acid sequence of SEQ ID NO:629 and a VL2 comprising the amino acid sequence of SEQ ID NO:1.

[0410] In some embodiments, the bispecific antibody EIP0820 comprises H2 comprising the amino acid sequence of SEQ ID NO:621 and L2 comprising the amino acid sequence of SEQ ID NO:67.

[0411] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions set forth above, the bispecific antibody EIP0820 comprises a VH1 having the amino acid sequence of SEQ ID NO:30. CDR1 , VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 and VL1 having the amino acid sequence of SEQ ID NO: 42. CDR1 , VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 and a VL1 comprising:

[0412] In some embodiments, the bispecific antibody EIP0820 comprises a VH1 having the amino acid sequence of SEQ ID NO: 17 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0820 comprises an H1 having the amino acid sequence of SEQ ID NO: 622 and an L1 having the amino acid sequence of SEQ ID NO: 69.

[0413] In some embodiments, an exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of the present disclosure comprises a CH1 H3 and / or CH2 H3 In some embodiments, the exemplary bispecific antibody comprises an amino acid at position 446 (EU numbering) and / or at position 447 (EU numbering) of H3 and / or CH2 H3 In some embodiments, the exemplary bispecific antibody comprises an amino acid at position 446 (EU numbering) of H3 and / or CH2 H3 If the bispecific antibody contains an amino acid at position 446 (EU numbering) of H3and / or an amino acid at position 447 (EU numbering) of CH2H3. H3 and / or CH2 H3 where the amino acid at position 447 (EU numbering) is K.

[0414] In some embodiments, an exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of the present disclosure comprises a CH1 H3 or CH2 H3 In some embodiments, the bispecific antibody comprises an amino acid at position 446 (EU numbering) of H3 and CH2 H3 In some embodiments, the bispecific antibody comprises an amino acid at position 446 (EU numbering) of H3 In some embodiments, the bispecific antibody comprises an amino acid at position 446 (EU numbering) of CH2 H3 Contains the amino acid at position 446 (EU numbering).

[0415] In some embodiments, the bispecific antibody is H3 and / or CH2 H3 In some embodiments, the bispecific antibody comprises an amino acid at position 446 (EU numbering) of H3 In some embodiments, the bispecific antibody comprises an amino acid at position 446 (EU numbering) of H3 In some embodiments, the bispecific antibody comprises an amino acid at position 446 (EU numbering) of H3 and CH2 H3 If it contains an amino acid at position 446 (EU numbering), CH1 H3 and CH2 H3 The amino acid at position 446 (EU numbering) is G.

[0416] In some embodiments, an exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody is H3and / or CH2 H3 In some embodiments, the bispecific antibody comprises an amino acid at position 447 (EU numbering) of H3 or CH2 H3 In some embodiments, the bispecific antibody comprises an amino acid at position 447 (EU numbering) of H3 and CH2 H3 In some embodiments, the bispecific antibody comprises an amino acid at position 447 (EU numbering) of H3 In some embodiments, the bispecific antibody comprises an amino acid at position 447 (EU numbering) of CH2 H3 Contains the amino acid at position 447 (EU numbering).

[0417] In some embodiments, the CD3ε x ULBP2 / 5 / 6 bispecific antibody is H3 and / or CH2 H3 In some embodiments, the bispecific antibody comprises an amino acid at position 447 (EU numbering) of H3 In some embodiments, the bispecific antibody comprises an amino acid at position 447 (EU numbering) of H3 where the amino acid at position 447 (EU numbering) is K.

[0418] Fusion peptide

[0419] Provided herein are antibodies (e.g., monospecific or bispecific antibodies) having a fusion peptide fused to the N-terminus or C-terminus of a first heavy chain polypeptide or a second heavy chain polypeptide.

[0420] Critical to the initial T cell response is the ability of T cells to detect foreign and mutant proteins through the T cell receptor. This response, often referred to as signal 1 of T cell activation, occurs when the T cell receptor binds to a cell presenting a foreign or mutant protein fragment or antigen in a specific protein complex called the major histocompatibility complex I (MHCI). Activation of the T cell receptor both activates and autoregulates T cells by itself. Strong binding of the TCR to the MHCI complex results in chronic activation of the TCR. This form of signaling is associated with T cells that are reactive against self-antigens. T cells are programmed to inactivate when this form of activation occurs. T cells that weakly bind the TCR but are sufficient for activation may undergo acute signaling and remain active to differentiate into memory T cells. This is emerging as an important consideration in the design of T cell therapeutics.

[0421] Activating T cell cytokines, often referred to as signal 3, are important in the transition of T cells from a non-dividing to a rapidly dividing state or from one phenotypic state to another. T cell cytokine receptors bind cytokines produced by immune and non-immune cells and, depending on the state of the T cell at the time of receiving the cytokine and cytokine signal, can induce cell proliferation, maintain viability, or induce differentiation of the T cell into a specialized cell state suitable for sustained activation or inactivation following infection.

[0422] One example is the cytokine-mediated transition of naive cells, which can induce naive T cells to proliferate and promote T cell differentiation into memory T cells. Exemplary cytokines include, but are not limited to, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, and IL-21.

[0423] Activation of a costimulatory receptor called signal 2 results in context-specific cell-cell reinforcement of T activation. The most recognized form of costimulation occurs when T cells interact with activated antigen-presenting cells through the CD80 and CD86 ligands and the T cell costimulatory receptor CD28 found on APCs. These interactions can "prime" specific T cells armed with T cell receptors to respond to pathogens or cancer proteins.

[0424] Less recognized is the costimulation induced at sites of infection and malignant lesions. This includes costimulation acting through CD2 and NKG2D receptors in response to ligands such as CD58 and UL16 binding proteins (e.g., ULBP2 / 5 / 6) induced in immune and epithelial cells during viral infection. These signals result in enhanced T activation as well as confirmation that T cell lethal effector activity is targeted with single-cell precision. Although many costimulatory receptors have been discovered, the importance of each receptor in the specific context and the impact of simultaneous signaling of multiple costimulatory receptors remains largely unknown and is an area that has much to advance our understanding of T cell biology and the potential for developing novel tumor-targeted T cell therapeutics.

[0425] Costimulatory ligands include, but are not limited to, CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, or ICOSLG.

[0426] CD58 has the advantage over other costimulatory ligands in that it is the primary costimulatory pathway available at the tumor site, but this is because tumor-infiltrating T lymphocytes often lose expression of other costimulatory receptors, such as CD28, or because tumor cells are poorly immunogenic, meaning that tumor cells do not sufficiently activate T cells, limiting the potential of inducible costimulatory receptors, such as 41BB.

[0427] As discussed above, the anti-CD3ε antibodies of the present disclosure induce different levels of T cell receptor activation that alter T cell survival and cytokine production. Thus, fusion of the costimulatory ligand CD58 to an anti-CD3ε bispecific antibody provides integrated costimulatory T cell activation for optimal T cell activation.

[0428] In some embodiments, the bispecific antibody has a peptide fused to the N-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific antibody has a peptide fused to the C-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific antibody has a polypeptide fused to the N-terminus of the second heavy chain polypeptide (H2). In some embodiments, the bispecific antibody has a peptide fused to the C-terminus of the second heavy chain polypeptide (H2). Exemplary peptides include, but are not limited to, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 or portions thereof. Exemplary peptides include, but are not limited to, CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, ICOSLG or portions thereof. Exemplary peptide sequences fused to bispecific antibodies include, but are not limited to, those listed in Table 15.1.

[0429] [Table 18]

[0430] In some embodiments, a CD3xULBP2 / 5 / 6 bispecific antibody of the invention has a CD58 fusion peptide comprising any one of the CD58 sequences in Table 15.2. In some embodiments, a CD3xULBP2 / 5 / 6 bispecific antibody of the invention has a CD58 fusion peptide comprising any one of SEQ ID NOs: 624-628. [Table 19]

[0431] In some embodiments, the polypeptide is directly fused to the bispecific antibody. In some embodiments, the polypeptide is indirectly fused via a linker. In some embodiments, the bispecific antibody fused to the peptide comprises a linker sequence. Exemplary linker sequences include, but are not limited to, those listed in Table 16.1.

[0432] [Table 20]

[0433] In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the invention has a CD58 fusion peptide (SEQ ID NO: 49) indirectly fused at the C-terminus of the first heavy chain polypeptide (H1) using linker 1 (SEQ ID NO: 52). In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the invention has a CD58v fusion peptide indirectly fused at the C-terminus of the first heavy chain polypeptide (H1) using linker 1 (SEQ ID NO: 52). * In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the invention has an IL-7 fusion peptide (SEQ ID NO: 51) indirectly fused at the C-terminus of the first heavy chain polypeptide (H1) using linker 1 (SEQ ID NO: 52).

[0434] In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the invention has a CD58 fusion peptide (SEQ ID NO: 49) indirectly fused at the C-terminus of the first heavy chain polypeptide (H1) using linker 2 (SEQ ID NO: 53). In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the invention has a CD58v fusion peptide indirectly fused at the C-terminus of the first heavy chain polypeptide (H1) using linker 2 (SEQ ID NO: 53). *In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the invention has an IL-7 fusion peptide (SEQ ID NO: 51) indirectly fused at the C-terminus of the first heavy chain polypeptide (H1) using linker 2 (SEQ ID NO: 53).

[0435] In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the invention has a CD58 fusion peptide (SEQ ID NO: 49) indirectly fused at the C-terminus of the first heavy chain polypeptide (H1) using linker 3 (SEQ ID NO: 54). In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the invention has a CD58v fusion peptide indirectly fused at the C-terminus of the first heavy chain polypeptide (H1) using linker 3 (SEQ ID NO: 54). * In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the invention has an IL-7 fusion peptide (SEQ ID NO: 51) indirectly fused at the C-terminus of the first heavy chain polypeptide (H1) using linker 3 (SEQ ID NO: 54).

[0436] In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the invention has a hinge sequence comprising any one of the linker sequences in Table 16.2.

[0437] In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the present invention has a linker sequence comprising any one of SEQ ID NOs: 530-552. [Table 21]

[0438] In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the invention has a linker sequence comprising any one of the linker sequences in Table 16.3.

[0439] In some embodiments, the CD3xULBP2 / 5 / 6 bispecific antibody of the present invention has a linker sequence comprising any one of SEQ ID NOs: 553-606. [Table 22-1] [Table 22-2]

[0440] CD58, CD58v * Exemplary CD3xULBP2 / 5 / 6 bispecific antibodies with IL-7 fusions are shown in Tables 17 and 18.

[0441] [Table 23]

[0442] [Table 24-1] [Table 24-2] [Table 24-3] [Table 24-4] [Table 24-5] [Table 24-6] [Table 24-7] [Table 24-8] [Table 24-9]

Table 24-10

Table 24-11

Table 24-12

Table 24-13

Table 24-14

Table 24-15

Table 24-16

Table 24-17

Table 24-18

Table 24-19

Table 24-20

Table 24-21

Table 24-22

Table 24-23

Table 24-24

Table 24-25

Table 24-26

Table 24-27

Table 24-28

Table 24-29

[0443]

Table 25-1

Table 25-2

Table 25-3

Table 25-4

Table 25-5

Table 25-6

Table 25-7

Table 25-8

Table 25-9

Table 25-10

Table 25-11

Table 25-12

Table 25-13

Table 25-14

Table 25-15

Table 25-16

Table 25-17

Table 25-18

Table 25-19

Table 25-20

Table 25-21

[0444] In some embodiments, the bispecific antibody EIP0373 comprises an L2 comprising the amino acid sequence of SEQ ID NO: 450, an H2 comprising the amino acid sequence of SEQ ID NO: 451, an L1 comprising the amino acid sequence of SEQ ID NO: 452, and an H1 comprising the amino acid sequence of SEQ ID NO: 453.

[0445] In some embodiments, the bispecific antibody EIP0535 comprises an L2 comprising the amino acid sequence of SEQ ID NO: 454, an H2 comprising the amino acid sequence of SEQ ID NO: 455, an L1 comprising the amino acid sequence of SEQ ID NO: 456, and an H1 comprising the amino acid sequence of SEQ ID NO: 457.

[0446] In some embodiments, the bispecific antibody EIP0506 comprises an L2 comprising the amino acid sequence of SEQ ID NO:458, an H2 comprising the amino acid sequence of SEQ ID NO:459, an L1 comprising the amino acid sequence of SEQ ID NO:460, and an H1 comprising the amino acid sequence of SEQ ID NO:461.

[0447] In some embodiments, the bispecific antibody EIP0534 comprises an L2 comprising the amino acid sequence of SEQ ID NO: 462, an H2 comprising the amino acid sequence of SEQ ID NO: 463, an L1 comprising the amino acid sequence of SEQ ID NO: 464, and an H1 comprising the amino acid sequence of SEQ ID NO: 465.

[0448] In some embodiments, the bispecific antibody EIP0702 comprises an L2 comprising the amino acid sequence of SEQ ID NO:466, an H2 comprising the amino acid sequence of SEQ ID NO:467, an L1 comprising the amino acid sequence of SEQ ID NO:468, and an H1 comprising the amino acid sequence of SEQ ID NO:469.

[0449] In some embodiments, the bispecific antibody EIP0703 comprises an L2 comprising the amino acid sequence of SEQ ID NO: 470, an H2 comprising the amino acid sequence of SEQ ID NO: 471, an L1 comprising the amino acid sequence of SEQ ID NO: 472, and an H1 comprising the amino acid sequence of SEQ ID NO: 473.

[0450] In some embodiments, the bispecific antibody EIP0765 comprises an L2 comprising the amino acid sequence of SEQ ID NO:474, an H2 comprising the amino acid sequence of SEQ ID NO:475, an L1 comprising the amino acid sequence of SEQ ID NO:476, and an H1 comprising the amino acid sequence of SEQ ID NO:477.

[0451] In some embodiments, the bispecific antibody EIP0766 comprises an L2 comprising the amino acid sequence of SEQ ID NO:478, an H2 comprising the amino acid sequence of SEQ ID NO:479, an L1 comprising the amino acid sequence of SEQ ID NO:480, and an H1 comprising the amino acid sequence of SEQ ID NO:481.

[0452] In some embodiments, the bispecific antibody EIP0990 comprises an L2 comprising the amino acid sequence of SEQ ID NO: 482, an H2 comprising the amino acid sequence of SEQ ID NO: 483, an L1 comprising the amino acid sequence of SEQ ID NO: 484, and an H1 comprising the amino acid sequence of SEQ ID NO: 485.

[0453] In some embodiments, the bispecific antibody EIP0991 comprises an L2 comprising the amino acid sequence of SEQ ID NO:486, an H2 comprising the amino acid sequence of SEQ ID NO:487, an L1 comprising the amino acid sequence of SEQ ID NO:488, and an H1 comprising the amino acid sequence of SEQ ID NO:489.

[0454] In some embodiments, the bispecific antibody EIP0991 comprises an L2 comprising the amino acid sequence of SEQ ID NO:486, an H2 comprising the amino acid sequence of SEQ ID NO:487, an L1 comprising the amino acid sequence of SEQ ID NO:488, and an H1 comprising the amino acid sequence of SEQ ID NO:489.

[0455] In some embodiments, the bispecific antibody EIP0992 comprises an L2 comprising the amino acid sequence of SEQ ID NO:490, an H2 comprising the amino acid sequence of SEQ ID NO:491, an L1 comprising the amino acid sequence of SEQ ID NO:492, and an H1 comprising the amino acid sequence of SEQ ID NO:493.

[0456] In some embodiments, the bispecific antibody EIP0993 comprises an L2 comprising the amino acid sequence of SEQ ID NO:494, an H2 comprising the amino acid sequence of SEQ ID NO:495, an L1 comprising the amino acid sequence of SEQ ID NO:496, and an H1 comprising the amino acid sequence of SEQ ID NO:497.

[0457] In some embodiments, the bispecific antibody EIP0869 comprises an L2 comprising the amino acid sequence of SEQ ID NO:498, an H2 comprising the amino acid sequence of SEQ ID NO:499, an L1 comprising the amino acid sequence of SEQ ID NO:500, and an H1 comprising the amino acid sequence of SEQ ID NO:501.

[0458] In some embodiments, the bispecific antibody EIP0870 comprises an L2 comprising the amino acid sequence of SEQ ID NO:502, an H2 comprising the amino acid sequence of SEQ ID NO:503, an L1 comprising the amino acid sequence of SEQ ID NO:504, and an H1 comprising the amino acid sequence of SEQ ID NO:505.

[0459] In some embodiments, the bispecific antibody EIP0871 comprises an L2 comprising the amino acid sequence of SEQ ID NO:506, an H2 comprising the amino acid sequence of SEQ ID NO:507, an L1 comprising the amino acid sequence of SEQ ID NO:508, and an H1 comprising the amino acid sequence of SEQ ID NO:509.

[0460] In some embodiments, the bispecific antibody EIP0872 comprises an L2 comprising the amino acid sequence of SEQ ID NO:510, an H2 comprising the amino acid sequence of SEQ ID NO:511, an L1 comprising the amino acid sequence of SEQ ID NO:512, and an H1 comprising the amino acid sequence of SEQ ID NO:513.

[0461] In some embodiments, the bispecific antibody EIP0546 comprises an L2 comprising the amino acid sequence of SEQ ID NO:514, an H2 comprising the amino acid sequence of SEQ ID NO:515, an L1 comprising the amino acid sequence of SEQ ID NO:516, and an H1 comprising the amino acid sequence of SEQ ID NO:517.

[0462] In some embodiments, the bispecific antibody EIP0607 comprises an L2 comprising the amino acid sequence of SEQ ID NO:518, an H2 comprising the amino acid sequence of SEQ ID NO:519, an L1 comprising the amino acid sequence of SEQ ID NO:520, and an H1 comprising the amino acid sequence of SEQ ID NO:521.

[0463] In some embodiments, the bispecific antibody EIP0614 comprises an L2 comprising the amino acid sequence of SEQ ID NO:522, an H2 comprising the amino acid sequence of SEQ ID NO:523, an L1 comprising the amino acid sequence of SEQ ID NO:524, and an H1 comprising the amino acid sequence of SEQ ID NO:525.

[0464] [Table 26-1] [Table 26-2] [Table 26-3] [Table 26-4] [Table 26-5] [Table 26-6] [Table 26-7]

[0465] [Table 27]

[0466] Production Method

[0467] Various procedures known in the art can be used to produce polyclonal or monoclonal antibodies directed against a given target, such as ULBP2 / 5 / 6, a disease-associated antigen or other target, or against a derivative, fragment, analog homolog or ortholog thereof (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0468] Antibodies are purified by well-known techniques such as affinity chromatography using Protein A or Protein G, which yields primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen or epitope that is the target of the desired immunoglobulin may be immobilized on a column to purify immune-specific antibodies by immunoaffinity chromatography. Purification of immunoglobulins is described, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0469] In some embodiments, the antibody of the present invention is a monoclonal antibody. Monoclonal antibodies are generated, for example, using the procedures described in the examples provided herein. Antibodies are also generated, for example, by immunizing BALB / c mice with combinations of cell transfectants that express high levels of a given target on their surface. Hybridomas resulting from myeloma / B cell fusions are then screened for reactivity against the selected target.

[0470] Monoclonal antibodies are prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

[0471] The immunizing agent typically includes a protein antigen, a fragment thereof, or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, and human origin. Usually, a rat or mouse myeloma cell line is used. The hybridoma cells can be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent growth of HGPRT-deficient cells.

[0472] Preferred immortalized cell lines are those that fuse efficiently, support stable high-level antibody expression by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which are available, for example, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines for the production of monoclonal antibodies have also been described (see Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63).

[0473] The culture medium in which the hybridoma cells were cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity for the target antigen.

[0474] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0475] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0476] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). The hybridoma cells of the invention are a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, that do not otherwise produce immunoglobulin proteins, to achieve synthesis of the monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by substituting coding sequences for human heavy and light chain constant domains for the homologous mouse sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368,812-13 (1994)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides may be substituted for the constant domains of an antibody of the invention, or may be substituted for the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0477] The monoclonal antibodies of the present invention include humanized or human antibodies. These antibodies are suitable for administration to humans without generating an immune response by the human to the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab') of antibodies) that are composed primarily of human immunoglobulin sequences and contain minimal sequence derived from non-human immunoglobulin. 2or other antigen-binding subsequences). Humanization can be accomplished, for example, by substituting rodent CDR or CDR sequences for the corresponding sequences of a human antibody following the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). (See U.S. Patent No. 5,225,539). Optionally, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies also comprise, for example, residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0478] A fully human antibody is an antibody molecule in which the entire sequences of both the light and heavy chains, including the CDRs, arise from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Monoclonal antibodies can be prepared by using trioma technology, human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), and EBV hybridoma technology to produce monoclonal antibodies (see Cole, et al., 1985 In:Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Monoclonal antibodies are available and can be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see Cole, et al., 1985 In:Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0479] Moreover, additional techniques can be used to produce human antibodies, including phage display libraries (see Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This technique is described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as in Marks et al., Bio / Technology 10, 779-783 (1992), Lonberg et al., Nature 368 856-859 (1994), Morrison, Nature 368, 812-13 (1994), Fishwild et al., Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13, 1997. 65-93(1995).

[0480] Human antibodies can also be produced using transgenic non-human animals that have been modified to produce fully human antibodies in response to antigen challenge rather than the animal's endogenous antibodies (see PCT Publication WO94 / 02602). Endogenous genes encoding immunoglobulin heavy and light chains in the non-human host are disabled, and active loci encoding human immunoglobulin heavy and light chains are inserted into the host's genome. Human genes are incorporated, for example, using yeast artificial chromosomes that contain the necessary human DNA segments. Intermediate transgenic animals containing less than the total number of modifications are then cross-bred to obtain animals that provide all the desired modifications as progeny. One example of such a non-human animal is the mouse called Xenomouse™, disclosed in PCT Publications WO96 / 33735 and WO96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from the animal following immunization with an immunogen of interest, e.g., as a polyclonal antibody preparation, or alternatively, from immortalized B cells derived from the animal, such as hybridomas that produce monoclonal antibodies. Additionally, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly, or further modified to obtain antibody analogs, e.g., single chain Fv (scFv) molecules.

[0481] One example of a method for producing a non-human host, exemplified as a mouse, that does not express endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. This can be accomplished by a method that includes deleting J segment genes from at least one endogenous heavy chain locus in embryonic stem cells to prevent rearrangement of the locus and prevent the formation of rearranged immunoglobulin heavy chain locus transcripts, the deletion being effected by a targeting vector containing a gene encoding a selectable marker, and producing a transgenic mouse from the embryonic stem cells whose somatic and germ cells contain the gene encoding the selectable marker.

[0482] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. The method involves introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses the antibody containing the heavy and light chains.

[0483] In a further improvement of this procedure, methods for identifying clinically significant epitopes in immunogens and correlative methods for selecting antibodies that specifically bind to the significant epitopes with high affinity are disclosed in PCT Publication WO 99 / 53049.

[0484] The antibody can be expressed by a vector containing a DNA segment encoding the single chain antibody described above.

[0485] These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO93 / 64701, which have a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in PCT / US95 / 02140 (WO95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phages, etc. Vectors may be chromosomal, non-chromosomal, or synthetic.

[0486] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney Murine Leukemia Virus. DNA viral vectors are preferred. These vectors include pox vectors, such as orthopox or avipox vectors, herpes virus vectors, such as type I herpes simplex virus (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI, et al., Proc Natl. Acad. Sci USA 87:1149 (1990)), adenovirus vectors (LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995)), and adeno-associated viral vectors (Kaplitt, MGet al., Nat. Genet. 8:148 (1994)).

[0487] Poxvirus vectors introduce genes into the cytoplasm of cells. Avipoxvirus vectors provide very short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into neural cells. The expression period of adenovirus vectors (approximately 2 months) is shorter than that of adeno-associated virus (approximately 4 months), which is shorter than that of HSV vectors. The particular vector selected depends on the target cell and the condition to be treated. The introduction can be performed by standard techniques, such as infection, transfection, transduction, or transformation. Examples of modes of gene transfer include, for example, naked DNA, (Ca)2 (PO 4 ) 3 These include precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0488] Vectors can be used to target essentially any desired target cell. For example, stereotactic injection can be used to direct vectors (e.g., adenovirus, HSV) to the desired location. In addition, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. A method based on total body flow, called convection, has also proven effective in delivering large molecules to large areas of the brain and can be useful for delivering vectors to target cells (see Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal and subcutaneous injection, as well as oral or other known routes of administration.

[0489] A bispecific antibody is an antibody that has binding specificities for at least two different antigens. In the present case, one of the binding specificities is for a first target, such as CD3ε or a fragment thereof. The second binding target is a disease-associated antigen, such as ULBP2 / 5 / 6, or a fragment thereof.

[0490] Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because the assortment of immunoglobulin heavy and light chains is random, these hybridomas (quadromas) produce a mixture of 10 possible different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually achieved by an affinity chromatography step. Similar procedures are disclosed in WO 93 / 08829 published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0491] The bispecific and / or monovalent antibodies of the invention can be produced using any of a variety of techniques recognized in the art, including those disclosed in co-pending application WO2012 / 023053, filed August 16, 2011, the contents of which are incorporated herein by reference in their entirety. The method described in WO2012 / 023053 produces bispecific antibodies that are identical in structure to human immunoglobulins. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant kappa domain, and a second light chain variable region fused to a constant lambda domain. Each binding site exhibits a different antigen specificity provided by both the heavy and light chains. The light chain variable region can be of the lambda or kappa family, and is preferably fused to the lambda and kappa constant domains, respectively. This is preferred to avoid the generation of non-natural polypeptide junctions. However, it is also possible to obtain bispecific antibodies of the invention by fusing a kappa light chain variable domain to a constant lambda domain for the first specificity and a lambda light chain variable domain to a constant kappa domain for the second specificity. The bispecific antibodies described in WO2012 / 023053 are called IgG κλ antibodies or "κλ bodies" and are a new fully human bispecific IgG format. This κλ body format allows affinity purification of bispecific antibodies that are indistinguishable from standard IgG molecules with properties indistinguishable from standard monoclonal antibodies and are therefore preferred compared to previous formats.

[0492] An essential step of the method is the identification of two antibody Fv regions (each composed of a variable light chain and a variable heavy chain domain) with different antigen specificities that share the same heavy chain variable domain. Numerous methods have been described for the production of monoclonal antibodies and fragments thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference). Fully human antibodies are antibody molecules in which both the light and heavy chain sequences, including CDRs 1 and 2, arise from human genes. The CDR3 region may be of human origin or designed by synthetic means. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". Human monoclonal antibodies can be prepared by using trioma techniques, human B cell hybridoma techniques (see Kozbor, et al., 1983 Immunol Today 4:72), and EBV hybridoma techniques to produce human monoclonal antibodies (see Cole, et al., 1985 In:Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies are available and can be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030), or by in vitro transformation of human B cells with Epstein-Barr virus (see Cole, et al., 1985 In:Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96).

[0493] Monoclonal antibodies are produced, for example, by immunizing animals with the target antigen or its immunogenic fragment, derivative or variant. Alternatively, animals are immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen, so that the target antigen is expressed and associated with the surface of the transfected cells. Various techniques for producing xenogeneic non-human animals are well known in the art. See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which are incorporated herein by reference in their entirety.

[0494] Alternatively, antibodies are obtained by screening libraries containing sequences of antibodies or antigen-binding domains for binding to the target antigen. The libraries are prepared, for example, in bacteriophages as protein or peptide fusions with bacteriophage coat proteins expressed on the surface of assembled phage particles and coding DNA sequences contained in the phage particles (i.e., "phage display libraries"). Alternatively, libraries can be prepared in yeast as protein or peptide fusions with cell wall proteins on the surface of yeast cells and coding DNA sequences contained in the yeast cells (i.e., "yeast display libraries").

[0495] Hybridomas resulting from myeloma / B cell fusion are then screened for reactivity to the target antigen. Monoclonal antibodies are prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to elicit lymphocytes that produce or can produce antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

[0496] Although it is not strictly impossible, it is highly unlikely that different antibodies that have the same heavy chain variable domain but are directed against different antigens are accidentally identified.In fact, in most cases, heavy chains contribute the most to antigen-binding surface and have the highest sequence variability.In particular, the CDR3 of heavy chains is the most diverse CDR in sequence, length and structure.Therefore, two antibodies that are specific to different antigens almost always have different heavy chain variable domains.

[0497] The method disclosed in co-pending application WO2012 / 023053 overcomes this limitation and greatly facilitates the isolation of antibodies with the same heavy chain variable domain by using an antibody library in which the diversity is restricted to the light chain variable domain because the heavy chain variable domains of all library members are the same. Such libraries are described, for example, in co-pending applications WO2010 / 135558 and WO2011 / 084255, each of which is incorporated herein by reference in its en...

Claims

1. A bispecific antibody fusion molecule comprising a bispecific antibody having a first antigen-binding domain that binds to CD3 and a second antigen-binding domain that binds to ULBP2 / 5 / 6, The first antigen-binding domain described above is (a) A first heavy chain variable region (VH1) comprising a complementarity-determining region 1 (CDRH1) containing the amino acid sequence of SEQ ID NO: 30, a CDRH2 containing the amino acid sequence of SEQ ID NO: 34, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 37; and (b) A first light chain variable region (VL1) comprising a complementarity-determining region 1 (CDRL1) containing the amino acid sequence of SEQ ID NO: 42, a CDRL2 containing the amino acid sequence of SEQ ID NO: 43, and a CDRL3 containing the amino acid sequence of SEQ ID NO:

45. Including; The second antigen-binding domain described above is (c) A second heavy chain variable region (VH2) comprising CDRH1 containing the amino acid sequence of SEQ ID NO: 5; CDRH2 containing the amino acid sequence of SEQ ID NO: 7; and CDRH3 containing the amino acid sequence of SEQ ID NO: 9; and (d) A second light chain variable region (VL2) comprising CDRL1 containing the amino acid sequence of SEQ ID NO: 10, CDRL2 containing the amino acid sequence of SEQ ID NO: 11, and CDRL3 containing the amino acid sequence of SEQ ID NO:

12. Includes, A bispecific antibody fusion molecule in which the aforementioned bispecific antibody is fused to a CD58 polypeptide.

2. The bispecific antibody fusion molecule according to Claim 1, wherein VH1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 17, and VL1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

22.

3. The bispecific antibody fusion molecule according to claim 1, wherein VH1 comprises the amino acid sequence of SEQ ID NO: 17 and VL1 comprises the amino acid sequence of SEQ ID NO:

22.

4. The bispecific antibody fusion molecule according to claim 1, wherein VH2 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 629, and VL2 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

5. The bispecific antibody fusion molecule according to claim 1, wherein VH2 comprises the amino acid sequence of SEQ ID NO: 629 and VL2 comprises the amino acid sequence of SEQ ID NO:

1.

6. The bispecific antibody fusion molecule according to claim 3, wherein VH2 comprises the amino acid sequence of SEQ ID NO: 629 and VL2 comprises the amino acid sequence of SEQ ID NO:

1.

7. The bispecific antibody fusion molecule according to claim 1, wherein the CD58 polypeptide comprises the amino acid sequence of SEQ ID NO: 49 or 50.

8. The bispecific antibody fusion molecule according to claim 1, wherein the CD58 polypeptide comprises the amino acid sequence of SEQ ID NO:

49.

9. The bispecific antibody fusion molecule according to claim 1, wherein the CD58 polypeptide is fused to the bispecific antibody by a linker.

10. The bispecific antibody fusion molecule according to claim 9, wherein the linker is a glycine-serine linker.

11. The bispecific antibody fusion molecule according to claim 9, wherein the linker comprises one amino acid sequence from SEQ ID NOs. 573 to 606.

12. The bispecific antibody fusion molecule according to claim 9, wherein the linker comprises the amino acid sequence of SEQ ID NO:

53.

13. The bispecific antibody fusion molecule according to claim 1, wherein the bispecific antibody comprises a first heavy chain (H1) containing VH1, a first light chain (L1) containing VL1, a second heavy chain (H2) containing VH2, and a second light chain (L2) containing VL2.

14. The bispecific antibody fusion molecule according to claim 13, wherein H1 comprises a first heavy chain constant region, L1 comprises a first light chain constant region, H2 comprises a second heavy chain constant region, and L2 comprises a second light chain constant region.

15. The bispecific antibody fusion molecule according to claim 1, wherein the CD58 polypeptide is linked to the C-terminus of H1.

16. The bispecific antibody fusion molecule according to claim 13, wherein H1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 622, and L1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

69.

17. The bispecific antibody fusion molecule according to claim 13, wherein H1 comprises the amino acid sequence of SEQ ID NO: 622 and L1 comprises the amino acid sequence of SEQ ID NO:

69.

18. The bispecific antibody fusion molecule according to claim 13, wherein H2 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 621, and L2 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

67.

19. The bispecific antibody fusion molecule according to claim 13, wherein H2 comprises the amino acid sequence of SEQ ID NO: 621 and L2 comprises the amino acid sequence of SEQ ID NO:

67.

20. The bispecific antibody fusion molecule according to claim 13, wherein H1 comprises the amino acid sequence of SEQ ID NO: 622, L1 comprises the amino acid sequence of SEQ ID NO: 69, H2 comprises the amino acid sequence of SEQ ID NO: 621, and L2 comprises the amino acid sequence of SEQ ID NO:

67.

21. A polynucleotide encoding a bispecific antibody fusion molecule according to any one of claims 1 to 20.

22. A vector comprising the polynucleotide described in claim 21.

23. A pharmaceutical composition comprising a bispecific antibody fusion molecule according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

24. A pharmaceutical for the treatment of cancer in a target area, comprising a bispecific antibody fusion molecule according to any one of claims 1 to 20.

25. The pharmaceutical agent according to claim 24, wherein the cancer is ULBP2, RAET1G (ULBP5), and / or RAET1L (ULBP6) positive cancer.

26. The pharmaceutical agent according to claim 24, wherein the cancer is a primary tumor, metastatic cancer, multidrug-resistant cancer, progressive tumor, or recurrent cancer.

27. The pharmaceutical product according to claim 24, wherein the cancer is a solid tumor.

28. The pharmaceutical product according to claim 24, wherein the cancer is bladder cancer, lung cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, melanoma, liver cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, skin cancer, or esophageal cancer.

29. The pharmaceutical product according to claim 24, wherein the treatment comprises administering a therapeutically effective amount of ligand or cytokine to the subject.

30. The pharmaceutical product according to claim 29, wherein the ligand is CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, ICOSLLG, MICA, MICB, ULBP1, ULBP2, ULBP3, RAET1G (ULBP5), and RAET1L (ULBP6); or the cytokine is IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, or IL-21, or an agonist antibody that binds to receptors for these ligands and cytokines.

31. The pharmaceutical agent according to claim 24, wherein the pharmaceutical agent prevents T cell exhaustion.

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